WO2023225488A1 - Peptides de translocation de barrière hémato-encéphalique et molécules associées et leurs méthodes d'utilisation - Google Patents

Peptides de translocation de barrière hémato-encéphalique et molécules associées et leurs méthodes d'utilisation Download PDF

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Publication number
WO2023225488A1
WO2023225488A1 PCT/US2023/067027 US2023067027W WO2023225488A1 WO 2023225488 A1 WO2023225488 A1 WO 2023225488A1 US 2023067027 W US2023067027 W US 2023067027W WO 2023225488 A1 WO2023225488 A1 WO 2023225488A1
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WIPO (PCT)
Prior art keywords
cyclotide
modified
peptide
seq
set forth
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PCT/US2023/067027
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English (en)
Inventor
Duncan Mcgregor
William Eldridge
Vaughn Smider
Charles MELANCON
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Enkefalos Biosciences, Inc.
Enkefalos Biosciences, Llc
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Publication of WO2023225488A1 publication Critical patent/WO2023225488A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present disclosure provides peptides able to bind a receptor that mediates receptor-mediating transcytosis (RMT) across the blood-brain barrier (BBB), as well as binding molecules that incorporate the peptides.
  • the present disclosure also provides conjugates, including fusion proteins, composed of the peptides or binding molecules and a therapeutic or diagnostic agent. In some embodiments, the conjugates are able to pass through the blood-brain barrier after being parenterally administered to allow for function of the therapeutic or diagnostic agent in the central nervous system.
  • the present disclosure also provides methods of making and using the provided peptides and molecules.
  • the blood-brain barrier performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of pharmacological agents to cerebral tissues, necessitating the use of vectors for their transit.
  • BBB permeability is frequently a rate-limiting factor for the penetration of drugs or peptides into the central nervous system (CNS) (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999); Bickel, U., Yoshikawa, T. & Pardridge, W. M. Adv. Drug Deliv. Rev. 46: 247-279 (2001).
  • the brain is shielded against potentially toxic substances by the BBB, which is formed by brain capillary endothelial cells that are closely sealed by tight junctions.
  • BBB brain capillary endothelial cells that are closely sealed by tight junctions.
  • brain capillaries possess few fenestrae and few endocytic vesicles, compared to the capillaries of other organs (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999)).
  • transferrin, lactoferrin and low-density lipoproteins which are taken up by receptor-mediated endocytosis (see Pardridge, W. M. J. Neurovirol. 5: 556-569 (1999); Tsuji, A.
  • a modified cyclotide comprising i) a peptide that binds to a blood-brain barrier trancytosis receptor (BBB-R) selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 Receptor Tyrosine Kinase 3 (ErbB3), leptin receptor (ObR), low-density lipoprotein receptor- related protein 1 (LRP-1), and receptor for advanced glycation-end products (RAGE), wherein said peptide has an amino acid sequence of 2 to 50 amino acid residues; and ii) a cyclotide scaffold comprising the peptide of i), wherein the modified cyclotide comprises the structure (I):
  • the BBB-R is human.
  • the cyclotide scaffold is selected from a plant cyclotide.
  • the cyclotide scaffold is of the Momordicae species.
  • the cyclotide scaffold is a Momordica cochinchinensis trypsin inhibitor.
  • the Momordica cochinchinensis trypsin inhibitor is MCoTI-I set forth in SEQ ID NO: 1, MCoTI- II set forth in SEQ ID NO: 2 or MCoTI-III set forth in SEQ ID NO: 3.
  • the sequence of said peptide replaces or substitutes one or more amino acids of one of the one or more loops of the cyclotide scaffold.
  • a modified cyclotide comprising a peptide that binds to a blood-brain barrier trancytosis receptor (BBB-R), wherein: the peptide is inserted into or replaces one or more amino acids of at least one loop of the cyclotide scaffold set forth in SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO: 3, and wherein the peptide is about 2 to 50 amino acid residues; and the BBB-R is selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 Receptor Tyrosine Kinase 3 (ErbB3), leptin receptor (ObR), low-density lipoprotein receptor-related protein 1 (LRP-1).
  • BBB-R blood-brain barrier trancytosis receptor
  • the cyclotide scaffold is set forth in SEQ ID NO:2.
  • the at least one loop is loop 1, loop 5 or loop 6, or is a combination thereof.
  • the at least one loop is loop 1.
  • the peptide is inserted into and replaces amino acids in only one loop of the cyclotide scaffold.
  • the only loop is loop 1.
  • the cyclotide scaffold is set forth in SEQ ID NO:2 and the peptide replaces loop 1 amino acids between cysteine 4 and cysteine 11 of SEQ ID NO:2.
  • a modified cyclotide comprising a peptide inserted into loop 1 to replace all amino acids between cysteine 4 and cysteine 11 of SEQ ID NO:2, wherein the peptide is 2 to 50 amino acid residues and binds to a blood-brain barrier trancytosis receptor (BBB-R) selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 Receptor Tyrosine Kinase 3 (ErbB3), leptin receptor (ObR), low-density lipoprotein receptor-related protein 1 (LRP-1).
  • BBB-R blood-brain barrier trancytosis receptor
  • the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 amino acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.
  • the peptide is 2 to 30 amino acids, such as 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids.
  • the peptide is 14 to 20 amino acids.
  • the peptide is 10 amino acids.
  • the peptide is 12 amino acids.
  • the BBB-R is expressed on brain endothelial cells.
  • the peptide has blood-brain barrier translocation activity.
  • the BBB-R is the transferrin receptor.
  • the peptide comprises the sequence set forth in any one of SEQ ID NOS: 26-34 and 49-54. In some embodiments, the peptide is set forth in any one of SEQ ID NOS: 26-34 and 49-54. In some of any of the provided embodiments, the peptide has the consensus motif set forth as xxxxxHxxSWGx (SEQ ID NOL: 177).
  • the modified cyclotide comprises the sequence set forth in any one of SEQ ID NOS: 72-80 and 95-100. In some embodiments, the modified cyclotide is set forth in any one of SEQ ID NOS: 72-80 and 95-100.
  • the peptide comprises the sequence forth in SEQ ID NO:26. In some embodiments, the peptide is set forth in SEQ ID NO:26. In some of any of the provided embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO:72. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:72. In some embodiments, the peptide comprises the sequence forth in SEQ ID NO:49. In some embodiments, the peptide is set forth in SEQ ID NO:49. In some embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO:95. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:95.
  • the peptide comprises a sequence with 1, 2, 3 or 4 amino acid substitution(s) compared to the sequence set forth in any one of SEQ ID NOS: 26-34 and 49-54.
  • the amino acid substitution(s) is substitution of an amino acid to another amino acid selected from histidine or an alanine.
  • the peptide comprises a sequence with 1, 2, 3 or 4 amino acid substitution(s) compared to the sequence set forth in of SEQ ID NO: 26.
  • the peptide comprises the sequence set forth in any one of SEQ ID NOS: 55 and 117-128.
  • the peptide is set forth in any one of SEQ ID NOS: 55 and 117-128.
  • the modified cyclotide comprises the sequence set forth in any one of SEQ ID NOS: 101 and 105-116. In some embodiments, the modified cyclotide is set forth in any one of SEQ ID NOS: 101 and 105-116.
  • the peptide comprises the sequence set forth in SEQ ID NO:55. In some embodiments, the peptide is set forth in SEQ ID NO:55. In some embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO: 101. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 101.
  • the BBB-R is the leptin receptor.
  • the peptide comprises the sequence set forth in any one of SEQ ID NOS: 10-23. In some of any of the provided embodiments, the peptide is set forth in any one of SEQ ID NOS: 10-23.
  • the modified cyclotide comprises the sequence set forth in any one of SEQ ID NOS: 56-69. In some embodiments, the modified cyclotide is set forth in any one of SEQ ID NOS: 56-69.
  • the peptide comprises a sequence with 1, 2, 3 or 4 amino acid substitution(s) compared to the sequence set forth in any one of SEQ ID NOS: 10- 23.
  • the amino acid substitution(s) is substitution of an amino acid to another amino acid selected from histidine or an alanine.
  • the BBB-R is ErbB3.
  • the peptide comprises the sequence set forth in SEQ ID NO:24 or 25. In some of any of the provided embodiments, the peptide is set forth in SEQ ID NO: 24 or 25.
  • the modified cyclotide comprises the sequence set forth SEQ ID NO: 70 or 71. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 70 or 71. In some of any of the provided embodiments, the peptide comprises a sequence with 1, 2, 3 or 4 amino acid substitution(s) compared to the sequence set forth in SEQ ID NO: 24 or 25. In some embodiments, the amino acid substitution(s) is substitution of an amino acid to another amino acid selected from histidine or an alanine.
  • the BBB-R is insulin-like growth factor type 1 receptor (IGFR).
  • IGFR insulin-like growth factor type 1 receptor
  • the peptide comprises the sequence set forth in SEQ ID NO:35 or 36 . In some of any of the provided embodiments, the peptide is set forth in SEQ ID NO: 35 or 36. In some of any of the provided embodiments, the peptide comprises the sequence set forth in SEQ ID NO:36. In some embodiments, the peptide is set forth in SEQ ID NO:36. In some of any of the provided embodiments, the modified cyclotide comprises the sequence set forth SEQ ID NO: 81 or 82.
  • the modified cyclotide is set forth in SEQ ID NO: 81 or 82. In some of any of the provided embodiments, the modified cycltoide comprises the sequence set forth in SEQ ID NO:82. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:82. In some of any of the provided embodiments, the peptide comprises a sequence with 1, 2, 3 or 4 amino acid substitution(s) compared to the sequence set forth in SEQ ID NO: 35 or 36. In some embodiments, the amino acid substitution(s) is substitution of an amino acid to another amino acid selected from histidine or an alanine.
  • the BBB-R is RAGE.
  • the peptide comprises the sequence set forth in SEQ ID NO:37 or 38. In some of any of the provided embodiments, the peptide is set forth in SEQ ID NO: 37 or 38.
  • the modified cyclotide comprises the sequence set forth SEQ ID NO: 83 or 84. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 83 or 84. In some of any of the provided embodiments, the peptide comprises a sequence with 1, 2, 3 or 4 amino acid substitution(s) compared to the sequence set forth in SEQ ID NO: 37 or 38. In some embodiments, the amino acid substitution(s) is to a histidine or an alanine.
  • the BBB-R is the 1 lipoprotein receptor-related protein 1 (LRP-1).
  • the peptide comprises the sequence set forth in any one of SEQ ID NOS: 39-48. In some of any of the provided embodiments, the peptide is set forth in any one of SEQ ID NOS: 39-48. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO:39. In some embodiments, the peptide is set forth in SEQ ID NO:39. In some embodiments, the peptide comprises the sequence set forth in SEQ ID NO:43. In some embodiments, the peptide is set forth in SEQ ID NO:43.
  • the peptide comprises the sequence set forth in SEQ ID NO:47. In some embodiments, the peptide is set forth in SEQ ID NO:47. In some of any of the provided embodiments, the modified cyclotide comprises the sequence set forth in any one of SEQ ID NOS: 85-94. In some embodiments, the modified cyclotide is set forth in any one of SEQ ID NOS: 85-94. In some embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO:85. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:85. In some embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO:89.
  • the modified cyclotide is set forth in SEQ ID NO:89. In some embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO:93. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:93. In some of any of the provided embodiments, the peptide comprises a sequence with 1, 2, 3 or 4 amino acid substitution(s) compared to the sequence set forth in any one of SEQ ID NOS: 39-48. In some embodiments, the amino acid substitution(s) is substitution of an amino acid to another amino acid selected from histidine or an alanine..
  • a peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 10-55 or 117-128, wherein the peptide is 6-50 amino acids in length and binds to a receptor involved in blood-brain barrier transcytosis (BBB-R).
  • BBB-R blood-brain barrier transcytosis
  • the peptide has blood-brain barrier translocation activity.
  • the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 amino acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.
  • the peptide is 2 to 30 amino acids, such as 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids.
  • the peptide is 14 to 20 amino acids.
  • the peptide is 10 amino acids.
  • the peptide is 12 amino acids.
  • a peptide consisting of the sequence set forth in any one of SEQ ID NOs: SEQ ID NOs: 10-55 or 117-128. In some of any of the provided embodiments, the peptide binds to a receptor involved in blood-brain barrier transcytosis.
  • the peptide is set forth in SEQ ID NO:26.
  • the peptide is set forth in SEQ ID NO: 49.
  • the peptide is set forth in SEQ ID NO: 55.
  • the peptide is set forth in SEQ ID NO:39.
  • the peptide is set forth in SEQ ID NO:43.
  • the peptide is set forth in SEQ ID NO:47.
  • the peptide is synthetic. In some of any of the provided embodiments, the peptide is isolated.
  • a binding molecule comprising a binding scaffold and any of the provided peptides.
  • the binding scaffold is a cyclotide.
  • the peptide is inserted into or replaces one or more amino acids of a loop of a cyclotide backbone.
  • the at least one loop is loop 1.
  • the binding scaffold is set forth in any one of SEQ ID NOS: 1-3.
  • the binding scaffold is set forth in SEQ ID NO:2.
  • nucleic acid molecule encoding any of the provided modified cyclotides or any of the provided binding molecules.
  • a vector comprising any of the provided nucleic acids.
  • the vector is an expression vector.
  • a host cell comprising any of the provided nucleic acid molecules or any of the provided vectors.
  • a method of producing a modified cyclotide or binding molecule comprising introducing the nucleic acid of claim 87 or the vector of claim 88 or claim 89 into a host cell and culturing the host cell under conditions to express the protein in the cell.
  • the method further includes purifying the protein from the cell.
  • a conjugate comprising any of the provided modified cyclotides, or any of the provided binding molecules, and a biological active agent.
  • the biologically active agent is a small molecule, a peptide or a protein.
  • the biologically active agent is a diagnostic agent or a therapeutic agent.
  • any of the provided conjugates is a fusion protein comprising the modified cyclotide operably linked to a biologically active agent that is a protein or peptide.
  • a fusion protein comprising any of the provided modified cyclotides or any of the provided binding molecules and a biologically active agent that is a protein or peptide.
  • the biologically active agent is an antibody.
  • the antibody is directed against an antigen selected from the group consisting of human epidermal growth factor receptor 2 (HER2), beta-secretase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6 and TNF-alpha.
  • HER2 human epidermal growth factor receptor 2
  • BACE1 beta-secretase 1
  • Abeta amyloid beta
  • EGFR epidermal growth factor receptor
  • Tau Tau
  • ApoE4 apolipoprotein E
  • the antibody is trastuzumab, adalimumab or aducanumab.
  • the biologically active agent is a growth factor or a hormone.
  • the biologically active agent is a growth factor and the growth factor is nerve growth factor (NGF) or Granulocyte colony-stimulating factor (GCSF).
  • the biologically active agent is an enzyme.
  • the enzyme is a ceramide degrading enzyme, a lipase, a hydrolase type enzyme or a sulfatase.
  • the enzyme is a ceramide degrading enzyme and the ceramide degrading enzyme is glucocerebrosidase, galactocerebrosidase or alpha galactosidase.
  • the enzyme is a glucocerebrosidase that has a sequence of amino acids that is at least 95% identical to the sequence set forth in SEQ ID NO: 144 or SEQ ID NO: 145.
  • the glucocerebrosidase is a variant that contains 1-5 amino acid substitutions compared to the sequence set forth in SEQ ID NO: 144 or SEQ ID NO: 145.
  • Non-limiting mutations include any as described herein.
  • the enzyme is a glucocerebrosidase that has the sequence of amino acids set forth in SEQ ID NO: 144.
  • the the enzyme is a glucocerebrosidase that has the sequence of amino acids set forth in SEQ ID NO: 145.
  • the enzyme is a lipase or a hydrolase type enzyme and the enzyme is sphinomyelinase, cerliponase or alpha glucosidase.
  • the conjugate or fusion protein comprises a single modified cyclotide. In some of any of the provided embodiments, the conjugate or fusion protein comprises 2, 3, or 4 modified cyclotides. In some of any of the provided embodiments, each modified cyclotide is the same. In some of any of the provided embodiments, each modified cyclotide is different.
  • the conjugate or fusion protein is monovalent for binding a BBB-R.
  • the conjugate is bivalent for binding to a BBB-R.
  • the conjugate or fusion protein comprises at least two different modified cyclotides that bind to different BBB-R.
  • the conjugate or fusion protein is bispecific for binding two different BBB-R.
  • the conjugate is bispecific for binding two different BBB-R and comprises at least two different modified cyclotides that each bind to a different BBB-R.
  • the modified cyclotide is linked to the biologically active agent via a linker.
  • the linker is at least 10 amino acids in length. In some embodiments, the linker is at least 15 amino acids in length. In some embodiments, the linker is 10 to 20 amino acids in length. In some of any of the provided embodiments, the linker is a flexible peptide linker. In som embodiments, the peptide linker comprises the sequence GGGGS (SEQ ID NO: 148), (GGGGS)2 (SEQ ID NO: 154) or (GGGGS)3 (SEQ ID NO: 154). In some of any of the provided embodiments, the linker is set forth in SEQ ID NO: 104.
  • the linker is a cleavable linker comprising an endosome-specific protease cleavage site.
  • the endosome-specific protease cleavage site is a cathepsin cleavage site.
  • the cathepsin cleavage site is a cathepsin B cleavage site.
  • the linker comprises the sequence set forth in SEQ ID NO: 133.
  • composition comprising any of the provided conjugates or fusion proteins and a pharmaceutical carrier.
  • a method for transporting a biologically active agent across a blood brain barrier of an individual comprising administering any of the provided conjugates or fusion proteins or any of the provided pharmaceutical compositions to an individualin need thereof.
  • the individual is a mammal. In some embodiments, the individual is a human.
  • the individual such as a mammal (e..g, a human) has a neurological disease.
  • said neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson's disease, stroke, a brain tumor and a brain metastasis.
  • said neurological disease is a congenital disease that is selected from the group consisting of Austen Disease, Canavan Disease, Gaucher’s Disease, Hunter Syndrome, Hurler-Scheie Syndrome, Jansky Bielschowsky Disease, Krabbe Disease, LCAT Deficiency, Lowe Syndrome, Maroteaux-Lamy Syndrome, Morquio Syndrome A, Morquio Syndrome B, Sanfilippo Syndrome A, Sanfilippo Syndrome B, Sanfilippo Syndrome C, Sanfilippo Syndrome D, Spinal Muscular Atrophy, Tay Sachs Disease, and Walker-Warburg Syndrome.
  • a method for treating a patient having a neurological disease comprising administering any of the provided conjugates or any of the provided pharmaceutical compositions to said patient.
  • a method for diagnosing a neurological disease in a patient in need thereof comprising administering any of the provided conjugates or fusion proteins or any of the provided pharmaceutical compositions to said patient and wherein said conjugate comprises a radiolabel.
  • said neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson's disease, stroke, a brain tumor and a brain metastasis.
  • said neurological disease is a congenital disease that is selected from the group consisting of Austen Disease, Canavan Disease, Gaucher’s Disease, Hunter Syndrome, Hurler-Scheie Syndrome, Jansky Bielschowsky Disease, Krabbe Disease, LCAT Deficiency, Lowe Syndrome, Maroteaux-Lamy Syndrome, Morquio Syndrome A, Morquio Syndrome B, Sanfilippo Syndrome A, Sanfilippo Syndrome B, Sanfilippo Syndrome C, Sanfilippo Syndrome D, Spinal Muscular Atrophy, Tay Sachs Disease, and Walker-Warburg Syndrome.
  • any of the compositions provided herein for use in the treatment of a neurological disease are also provided. Also provided are use of any of the provided pharmaceutical compositions in the manufacture of a medicament for use in the treatment of a neurological disease. Also provided are any of the provided compositions provided herein for use in the diagnosis of a neurological disease. Also provided herein are use of any of the provided pharmaceutical compositions in the manufacture of a medicament for use in the diagnosis of a neurological condition.
  • said neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson's disease, stroke, a brain tumor and a brain metastasis.
  • said neurological disease is a congenital disease that is selected from the group consisting of Austen Disease, Canavan Disease, Gaucher’s Disease, Hunter Syndrome, Hurler-Scheie Syndrome, Jansky Bielschowsky Disease, Krabbe Disease, LCAT Deficiency, Lowe Syndrome, Maroteaux-Lamy Syndrome, Morquio Syndrome A, Morquio Syndrome B, Sanfilippo Syndrome A, Sanfilippo Syndrome B, Sanfilippo Syndrome C, Sanfilippo Syndrome D, Spinal Muscular Atrophy, Tay Sachs Disease, and Walker-Warburg Syndrome.
  • FIGS. 1A-1D depict exemplary configurations of provided antibody fusion proteins composed of an antibody and at least one modified cyclotide including: a bivalent molecule (FIG. 1A), a bispecific molecule (FIG. IB), a monovalent molecule in which a hole heavy chain is modified with the modified cyclotide (FIG. 1C), and a monovalent molecule in which a knob heavy chain is modified with the modified cyclotide (FIG. ID).
  • a bivalent molecule FIG. 1A
  • a bispecific molecule FIG. IB
  • FIG. 1C a monovalent molecule in which a hole heavy chain is modified with the modified cyclotide
  • FIG. ID monovalent molecule in which a knob heavy chain is modified with the modified cyclotide
  • FIG. 2 depicts a flow cytometry -based assay for internalization of fluorescently labeled adalimumab-CYC17 cyclotide fusion and adalimumab by transferrin receptorexpressing human cells.
  • FIG. 3 depicts ELISA results for binding of different adalimumab-cyclotide fusions for binding to transferrin receptor (TfR) coated on a microwell, in which the cyclotide portion was CYC17 or different mutants thereof containing amino acid substitution at the indicated positions (e.g. 5H, refers to substitution to a histidine (H) at position 5).
  • TfR transferrin receptor
  • FIG. 4 depicts binding of different cyclotide antibody fusion molecules to human transferrin receptor, including molecules set forth in FIGS. 1 A-1D.
  • the molecules include monovalent molecules in which the exemplary CYC 17 was fused to either the knob or hole chain of a heterodimeric aducanumab (Aduhelm) antibody, a bispecific fusion in which CYC17 (directed to TfR) was fused to the hole and CYC27 (directed to IGF1R) was fused to the knob of a heterodimeric aducanumab (Aduhelm) antibody, and a bivalent adalimumab (Humira) containing two copies of the CYC 17 cyclotide.
  • Aduhelm heterodimeric aducanumab
  • Humira bivalent adalimumab
  • FIG. 5 depicts expression of cyclotide trastuzumab antibody fusion molecules in supernatant from transfected host cells.
  • FIG. 6A depicts enzymatic activity of the GlcCSase-CYC17 fusion cyclotide in a glucocerebroside activity assay.
  • FIG. 6B depicts ELISA results for binding of the GlcCSase-CYC17 fusion cyclotide to the human transferrin receptor (hTfR) coated on a microwell.
  • FIG. 7 depicts ELISA results for binding of the NGF-CYC17 fusion cyclotide to the human transferrin receptor (hTfR) coated on a microwell.
  • FIG. 8A depicts ELISA results for binding of exemplary trastuzumab fusion cyclotides to human LPR1 coated on a microwell.
  • the fusion molecules included monovalent molecules in which exemplary cyclotides CYC30, CYC32, or CYC34 were fused to the knob chain of a heterodimeric trastuzumab (Herceptin®) antibody.
  • FIG. 8B depicts quantification results of in vivo cyclotide-mediated trastuzumab delivery into mouse brains at 24 hours compared to trastuzumab alone.
  • the fusion molecules included monovalent molecules in which exemplary cyclotides CYC26, CYC27, CYC28, CYC29, CYC30, CYC32, or CYC34 were fused to the knob chain of a heterodimeric trastuzumab (Herceptin®) antibody.
  • FIG. 9 depicts quantification results of in vivo cyclotide-mediated trastuzumab delivery into mouse brains at 24 hours compared to trastuzumab alone.
  • Two antibody- monovalent cyclotide fusion molecules, targeting human transferrin receptor (hTfR) were administered to mice and quantified from fresh brain homogenates.
  • the fusion molecules include monovalent molecules in which exemplary cyclotides CYC26 or CYC27 were fused to the knob chain of a heterodimeric trastuzumab (Herceptin®) antibody.
  • peptides that exhibit blood-brain barrier translocation activity (including the ability to mediate transcytosis to facilitate crossing of the BBB), such as via binding to a receptor involved in blood-brain barrier transcytosis.
  • the provided peptides can be inserted into a protein scaffold to provide for a larger binding molecule backbone or framework, which, in some aspects, may improve the stability or half-life of the peptide.
  • the scaffold is a a cyclotide backbone to provide for a modified cyclotide with improved or enhanced blood-brain barrier translocation activity and/or binding to a receptor involved in blood-brain barrier transcytosis.
  • the provided modified cyclotide molecules are non-naturally occurring and demonstrate particular advantage in crossing the blood-brain barrier.
  • the provided modified scaffolds into which is inserted a provided peptide, such as provided modified cyclotides can be used as carriers for attachment or linkage to other molecules, such as polypeptides, proteins and small molecules, including therapeutic agents or diagnositic agents, to delivery such other molecules across the bloodbrain barrier.
  • the provided modified cyclotides may be used to deliver therapeutic agents and other biological agents across the BBB for the treatment of neurological conditions.
  • a problem in delivery of certain therapeutic or other biological agents is that it is difficult to deliver biological agents administered parenterally, such as therapeutic agents and diagnostic agents, to the central nervous system (CNS) because passive transfer of substances from the capillaries to the brain is restricted.
  • CNS central nervous system
  • the capillaries that supply the blood to most of the brain tissues differ in that the endothelial cells forming their endothelium are mutually connected by tight intercellular junctions.
  • This system which restricts exchange of substances between the blood and the tissue fluid of the brain through the endothelium of capillaries in the brain, is called the blood-brain barrier or BBB.
  • the BBB thus imposes a problem in the access of therapeutic or other biological agents to the CNS because they cannot always pass through the BBB. For instance approximately only about 0.2% of an monoclonal antibody dose that is administered intravenously will reach the brain when the BBB is intact.
  • the provided embodiments address these problems.
  • the provided embodiments relate to novel peptides that exhibit binding to receptors involved in blood-brain barrier transcytosis. These peptides can be incorporated into a binding molecule scaffold to provide stability to the molecule and confer onto the molecule the ability to bind receptors that mediate RMT across the blood-brain barrier (BBB).
  • the binding molecule scaffold is a cyclotide, which is a cysteine-knot protein.
  • the ability to incorporate peptides into stable scaffolds, such as cyclotides provides for a highly developable platform involving small, highly stable peptides that express well as fusion proteins.
  • the provided embodiments also can be used to produce multi-domain molecules with the ability to target more than one transcytosis pathway.
  • Cysteine-knot microproteins are small peptides, typically consisting of about 30-40 amino acids, which can be found naturally as cyclic or linear forms, where the cyclic form has no free N- or C-terminal amino or carboxyl end. They have a defined structure based on three intramolecular disulfide bonds and a small triple stranded P- sheet (Craik et al., 2001, Toxicon 39, 43-60).
  • the cyclic proteins exhibit conserved cysteine residues defining a structure referred to herein as a "cysteine knot".
  • This family includes both naturally occurring cyclic molecules and their linear derivatives as well as linear molecules which have undergone cyclization. These molecules are useful as molecular framework structures having enhanced stability over less structured peptides(Colgrave and Craik, 2004, Biochemistry 43, 5965-5975). However, these molecules are not themselves capable of crossing the blood-brain barrier to any great extent, and cannot be used as neurotherapeutic agents themselves or act as carriers of therapeutic peptide or protein agents.
  • the main cyclotide features are a remarkable stability due to the cysteine knot, a small size making them readily accessible to chemical synthesis, and an excellent tolerance to sequence variations. Cyclotides therefore appear as appealing leads or scaffolds for peptide drug design.
  • the cyclotide scaffold is found in almost 30 different protein families among which conotoxins, spider toxins, squash inhibitors, agouti -related proteins and plant cyclotides are the most populated families. Cyclotides from plants in the Rubiaceae and Violaceae families are for the most part found to be head-to-tail cyclic peptides.
  • a cyclotide sequence is characterized as having a cysteine knot backbone moiety, in which the cysteine knot backbone comprises the structure (I): Loop6 Loopl Loop2 Loop3 Loop4 Loop5 Loop6 wherein Ci to Ce are cysteine residues; wherein each of Ci and C 4 , C 2 and Cs, and C 3 and Ce are connected by a disulfide bond to form a cysteine knot; wherein each X represents an amino acid residue in a loop, wherein said amino acid residues are the same or different; wherein d is about 1-2; and wherein a, b, c, e, and f, are the same or different, and are each any number from 3-10, and b, c, e, and f are each any number from 1 to 20.
  • the cysteine knot backbone comprises the structure (I): Loop6 Loopl Loop2 Loop3 Loop4 Loop5 Loop6 wherein Ci to Ce are cysteine residues; wherein each of Ci and C 4 , C 2 and
  • cyclotides in addition to stability of the molecule, cyclotides also exhibit features that indicate the molecules have a low immunogenicity risk. Although cyclotides originate as a non-human sequence, they are a heavily disulfide-bonded protein. Evidence indicates that non-human disulfide bonded peptides exhibit no detectable immunogenicity. For instance, Ziconitide is an FDA approved disulfide bonded non-human peptide analogous to cyclotides, and no anti-drug antibodies (AD As) were detectable, even after repeat dose, in preclinical mouse and rat immunogenicity testing (Skov M. et al. Int. J. Toxicology, 2007 26:411-421).
  • the provided molecules address these needs.
  • the provided modified cyclotide molecules are useful as a carrier microprotein peptides capable of crossing the BBB, and can be fused to a desired biological agent, such as a therapeutic molecule, for delivery to the brain.
  • the embodiments provided herein relate to a cyclotide molecular framework to generate modified cyclotides into which is inserted a peptide sequence to confer enhanced BBB translocation activity relative to the parental cyclotide.
  • the modified cyclotide is composed of a cysteine-knot backbone that has sufficient disulfide bonds or chemical equivalents thereof to confer a knotted topology on the three-dimensional structure of said cysteine-knot backbone and wherein at least one exposed amino acid residue such as on one or more beta turns and/or within one or more loops, is inserted or substituted (replaced) with a receptor-binding peptide relative to the naturally occurring cyclotide amino acid sequence.
  • the modified cyclotide has enhanced translocation behaviour compared with the unmodified parental cyclotide. In some embodiments, the modified cyclotide has the desired properties of high enzymatic stability and translocation, such that blood-brain barrier transfer of the modified cyclotide is feasible.
  • a modified cyclotide sequence provided herein may be defined as having a cysteine knot backbone moiety and a peptide that is a blood-brain barrier translocation moiety, said modified cyclotide comprising : i) a peptide having said bloodbrain barrier translocation activity, wherein said peptide has an amino acid sequence that is about 2 to 50 amino acid residues; and ii) a cysteine knot backbone grafted to said peptide of clause i), wherein said cysteine knot backbone comprises the structure (I): )
  • Ci to Ce are cysteine residues; wherein each of Ci and C 4 , C 2 and Cs, and C 3 and Ce are connected by a disulfide bond to form a cysteine knot; wherein each X represents an amino acid residue in a loop, wherein said amino acid residues are the same or different; wherein d is about 1-2; wherein one or more of loops 1, 2, 3, 5 or 6 have an amino acid sequence comprising the sequence of said peptide of clause i), wherein any loop comprising said sequence of said peptide of clause i) comprises 2 to about 50 amino acids, and wherein for any of loops 1, 2, 3, 5, or 6 that do not contain said sequence of said peptide of clause i), a, b, c, e, and f, are the same or different, and are each any number from 3-10, and b, c, e, and f are each any number from 1 to 20.
  • the provided modified cyclotide sequence may be either linear or cyclic.
  • the cysteine knot backbone is a cyclotide of the Momordicae species including the squash serine protease inhibitor family.
  • Exemplary cyclotides of the Mom ori dicase species include those set forth in SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3.
  • the peptide is inserted into or replaces one or more amino acids of at least one loop of the cyclotide scaffold set forth in SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO: 3.
  • the cyclotides provided herien are modified cyclotides compared to a natural or wildtype unmodified cyclotide, in which the modified cyclotide has one or more loops inserted or substituted by one or more amino acid sequences, e.g. a peptide of 2 to 50 amino acids.
  • the one or more amino acids e.g.
  • a peptide sequence of 2 to 50 amino acids, that is inserted or substituted into a loop of an unmodified or wildtype cyclotide is a blood-brain barrier translocation moiety that is able to bind to a receptor involved in trancytosis across the blood-brain barrier and/or that mediates blood-brain barrier translocation or permeation of the cyclotide.
  • the peptide is one that bind to a transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 Receptor Tyrosine Kinase 3 (ErbB3), leptin receptor (ObR), low-density lipoprotein receptor-related protein 1 (LRP-1), or receptor for advanced glycation end products (RAGE).
  • TrfR transferrin receptor
  • IGFR insulin-like growth factor type 1 receptor
  • ErbB3 Erb-B2 Receptor Tyrosine Kinase 3
  • ObR leptin receptor
  • LRP-1 low-density lipoprotein receptor-related protein 1
  • RAGE receptor for advanced glycation end products
  • the modified cyclotides of the invention incorporate sufficient amino acid structure to provide high enzymatic stability and good translocation or permeation behaviour.
  • the provided modified cyclotides can be used as novel carrier cyclotides to facilitate or mediate translocation of an attached polypeptid
  • the peptide-modified cyclotides may be conjugated to another agent or polypeptide, or in certain embodiments one or more modified cyclotides are incorporated into another polypeptide to form a synthetic chimeric (or fusion) polypeptide/protein.
  • the peptide of the modified cyclotides may be comprised within a loop region of a scaffold polypeptide.
  • the resultant modified scaffold can then itself be conjugated or comprised within a larger molecule.
  • cyclotides into which is inserted a receptor-binding peptide as provided herein may be enriched for cysteine residues.
  • the provided modified cyclotides exhibit the ability to traverse the blood-brain barrier, suitably rendering the modified cyclotide or any molecule comprising or conjugated thereto available to the brain.
  • the provided binding molecules may be used to deliver a biologically active agent, such as a peptide, polypeptide, protein or small molecule, across the blood-brain barrier.
  • a biologically active agent such as a peptide, polypeptide, protein or small molecule
  • the biologically active agent is a therapeutic agent, such as a therapeutic agent with therapeutic agonist or antagonist activity.
  • the biologically active agent is a diagnostic agent.
  • a provided modified cyclotide such as a linear or cyclic cyclotide, is operably linked to the N- or C-terminus of a biologically active agent, such as a peptide or protein moiety.
  • the linkage may be direct or indirect via a peptide linker.
  • the linkage provides a conjugate, e.g. fusion protein, of the modified cyclotide and the biologically active agent.
  • a conjugate, e.g. a fusion protein comprising a provided modified cyclotide operably linked to the N- or C-terminus of a biologically active agent, such as a peptide or protein moiety.
  • the biologically active agent is a therapeutic agent, such as an agent that is agonist or an antagonist.
  • the linkage or fusion of the biologically active agent with a provided modified cyclotide allows the translocation across the BBB and entry of the biologically active agent, such as a peptide or protein moiety.
  • such linkage allows the translocation across the BBB, and entry of a pharmacologically relevant dose of the biologically active agent, such as a peptide or protein moiety.
  • bispecific molecules that incorporate more than one receptor-binding molecule to thereby target more than one transcytosis pathway.
  • at least two different modified cyclotides can be linked to a biologically active agent to provide a multidomain protein that may further improve transcytosis capacity of the linked biological agent across the BBB.
  • 1, 2, 3 or 4 different modified cyclotides each modified with a different receptor-binding peptide may be linked or conjugated to a biological agent to permit engagement of one or multiple BBB transporter pathways.
  • peptides of 2-50 amino acids in length that bind a receptor involved in blood-brain barrier transcytosis In some embodiments, provided herein are peptides of 6-50 amino acids in length that bind a receptor involved in blood-brain barrier transcytosis. In some aspects, the provided peptides exhibit blood-brain barrier translocation activity. Also provided herein are binding molecules that incorporate any of the provided peptides into a binding molecule scaffold. In some embodiments, the binding molecule scaffold is a small cysteine-knot protein. In some embodiments, the cysteine-knot protein is a cyclotide.
  • cyclotides that are modified compared to a parental or native cyclotide backbone (also called a cysteine knot backbone), in which the provided modified cyclotides exhibit improved or enhanced ability to bind to a receptor involved in transcytosis across the blood-brain barrier (BBB; hereinafter “BBB trancytosis receptor”) and/or exhibit improved or enhanced blood-brain barrier translocation characteristics.
  • a parental or native cyclotide backbone also called a cysteine knot backbone
  • BBBB trancytosis receptor blood-brain barrier translocation characteristics
  • BBB-R BBB transcytosis receptor
  • the peptides are or may be inserted into the backbone of a binding molecule scaffold, such as a cysteine-knot scaffold, for example a cyclotide.
  • a binding molecule scaffold such as a cysteine-knot scaffold, for example a cyclotide.
  • Exemplary modified cyclotides containing a receptorbinding peptide are described in subsection I.B.
  • the BBB-R is expressed on brain endothelial cells.
  • the BBB-R is selected from the transferrin receptor (TrfR); the lactoferrin receptor (LtfR); the leptin receptor (LEP-R, or also known as OB-R); the receptor tyrosineprotein kinase (ErbB3), a insulin receptor, such as Insulin Receptor A (IRA), IGF-1 Receptor (IGF-1R), IGF-II Receptor (IGF-IIR), RXFP1, RXFP2, RXFP3 and RXFP4; low-density lipoprotein receptor-related protein 1 (LRP-1), the receptor for advanced glycation end products (RAGE); heparin binding EGF like growth factor receptor (HB EGFR); the intercellular adhesion molecule 1 (ICAM1), intercellular adhesion molecule 2 (ICAM2) or neural cell adhesion molecule (NCAM); or any other receptor
  • TrfR transferrin receptor
  • the BBB-R is a mammalian receptor. In some embodiments, the BBB-R is murine. In some embodiments, the BBB-R is human.
  • the BBB-R is selected from the human transferrin receptor (human TrfR); the human lactoferrin receptor (human LtFR); the human leptin receptor (human Ob-R); the human ErbB3 receptor, a human insulin receptor, such as Insulin Receptor A (IRA), IGF-1 Receptor (IGF-1R), IGF-II Receptor (IGF-IIR), RXFP1, RXFP2, RXFP3 and RXFP4; the human low-density lipoprotein receptor-related protein 1 (human LRP-1); the human receptor for advanced glycation end products (human RAGE); human heparin binding EGF like growth factor receptor (human HB EGFR); the human intercellular adhesion molecule 1 (human ICAM1), intercellular adhesion molecule 2 (human ICAM2) or neural cell adhesion molecule (human NCAM).
  • human TrfR human lactoferrin receptor
  • human Ob-R human leptin receptor
  • ErbB3 receptor a human insulin receptor
  • the BBB-R is Erb-B2 Receptor Tyrosine Kinase 3 (ErbB3), such as human ErbB3.
  • the BBB-R is TrfR, such as human TrfR.
  • the BBB-R is leptin receptor (ObR), such as human ObR.
  • the BBB-R is insuline-like growth factor type 1 receptor (IGFR), such as human IgFR.
  • the BBB-R is low-density lipoprotein-related protein 1 (LRP-1), such as human LRP-1.
  • the BBB-R is receptor for advanced glycation end products (RAGE), such as human RAGE.
  • the peptides are synthetic peptides. In some embodiments, the peptides are isolated peptides.
  • the peptide has an amino acid sequence that is about 2 to 50 amino acid residues in length. In some embodiments, the peptide is 2 to 50 amino acid residues. In some embodiments, the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 amino acids, 30 amino acids, 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino
  • the peptide is 2 to 30 amino acids, such as 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids.
  • the peptide is 10 to 25 amino acids. In some embodiments, the peptide is 10 amino acids. In some embodiments, the peptide is 11 amino acids. In some embodiments, the peptide is 12 amino acids. In some embodiments, the peptide is 13 amino acids. In some embodiments, the peptide is 14 amino acids. In some embodiments, the peptide is 15 amino acids. In some embodiments, the peptide is 16 amino acids. In some embodiments, the peptide is 17 amino acids. In some embodiments, the peptide is 18 amino acids. In some embodiments, the peptide is 19 amino acids. In some embodiments, the peptide is 20 amino acids. In some embodiments, the peptide is 21 amino acids. In some embodiments, the peptide is 22 amino acids. In some embodiments, the peptide is 23 amino acids. In some embodiments, the peptide is 24 amino acids. In some embodiments, the peptide is 25 amino acids.
  • the BBB-R antigen to be used for production of, or screening for, peptide may be a soluble form of or a portion thereof (e.g. the extracellular domain) containing the desired epitope.
  • the BBB-R target molecules may be isolated from natural sources or prepared by recombinant methods by procedures known in the art.
  • the purified target molecule can be attached to a suitable matrix such as agarose beads, acrylamide beads, glass beads, cellulose, various acrylic copolymers, hydroxyalkyl methacrylate gels, polyacrylic and polymethacrylic copolymers, nylon, neutral and ionic carriers, and the like.
  • Attachment of the target protein to the matrix may be accomplished by methods described in Methods in Enzymology, 44 1976, or by other means known in the art.
  • cells expressing BBB-R at their cell surface can be used to generate, or screen for, binding molecules.
  • binding molecules can be screened for membrane translocation activity can be
  • random peptides can be inserted into the backbone of a scaffold sequence, such as a cyclotide including any as described in Section I.B, and the scaffold library can be used to screen for binding to a BBB-R.
  • a scaffold sequence such as a cyclotide including any as described in Section I.B
  • Any known methods for generating libraries containing variant polynucleotides and/or polypeptides can be used with the provided methods and vectors to generate display libraries, e.g. phage display libraries, and to select binding proteins from the libraries.
  • the libraries can be used in screening assays to select binding proteins from the library for binding to a BBB-R.
  • libraries of variant binding molecules typically are screened using a display technique, such that there is a physical link between the individual molecules of the library (phenotype) and the genetic information encoding them (genotype).
  • display technique such that there is a physical link between the individual molecules of the library (phenotype) and the genetic information encoding them (genotype).
  • methods include, but are not limited to, cell display, including bacterial display, yeast display, mammalian display, phage display (Smith, G. P. (1985) Science 228: 1315-1317), mRNA display, ribosome display and DNA display.
  • a library of cyclotides each inserted with a random peptide is screened to identify individual members of the library that exhibit a desired biological activity such as binding to a BBB-R.
  • a method for screening for BBB-R binding includes the steps of: a) constructing a cyclotide sequence display library in which random peptides are inserted into the cyclotide scaffold (e.g. into a loop therein, e.g.
  • loop 1 b) expressing the sequence library in order to obtain expressed cyclotides, or polypeptides that comprise the cyclotides; c) selecting the expressed sequence library against a suitable purified BBB-R; d) recovering any cyclotide binding to the receptor; e) determining the sequence of the recovered cyclotide or peptide contained therein.
  • the display library is a phage display library of a plurality of modified cyclotide scaffold (or other binding molecule scaffold) in which each is inserted with a random peptide.
  • each modified cyclotide scaffold of the library is fused to a phage coat protein and displayed, usually on average as a single copy of each related polypeptide, on the surface of a phagemid particle containing DNA encoding that polypeptide.
  • These phagemid particles are then contacted with a BBB-R target and those particles having the highest affinity for the target are separated from those of lower affinity.
  • the higher affinity binders are then amplified by infection of a bacterial host and the competitive binding step is repeated. This process is reiterated until polypeptides of the desired affinity are obtained.
  • the provided methods include contacting any of the display libraries provided herein with a target molecule under conditions to allow binding of a display particle, e.g., a phagemid particle, to the target molecule.
  • the methods further include separating the display particles, e.g., the phagemid particles, that bind from those that do not, thereby selecting display particles, e.g., the phagemid particles, that include an antibody binding protein that binds to the target molecule.
  • the methods include sequencing the fusion gene in the selected particles to identify the antibody binding protein.
  • the BBB-R target molecule is contacted with the library of display particles, e.g., phagemid particles, under conditions suitable for binding of at least a portion of the display particles with the target molecules.
  • the conditions including pH, ionic strength, temperature and the like will mimic physiological conditions.
  • Exemplary “contacting” conditions may comprise incubation for 15 minutes to 4 hours, e.g. one hour, at 4°-37° C., e.g. at room temperature. However, these may be varied as appropriate depending on the nature of the interacting binding partners, etc.
  • the mixture can be subjected to gentle rocking, mixing, or rotation.
  • other appropriate reagents such as blocking agents to reduce nonspecific binding may be added.
  • the contacting conditions can be varied and adapted by a skilled person depending on the aim of the screening method.
  • the incubation temperature is, for example, room temperature or 37° C.
  • this may increase the possibility of identifying binders which are stable under these conditions, e.g., in the case of incubation at 37° C., are stable under conditions found in the human body.
  • Such a property might be extremely advantageous if one or both of the binding partners was a candidate to be used in some sort of therapeutic application, e.g. an antibody. Again such adaptations to the conditions are within the ambit of the skilled person
  • Binders having high affinity for the immobilized target molecule can be separated from those having a low affinity (and thus do not bind to the target) by washing. Binders can be dissociated from the immobilized target molecules by a variety of methods. These methods include competitive dissociation using the wild-type ligand, altering pH and/or ionic strength, and methods known in the art.
  • peptides can be screened for their activity to confer blood brain barrier translocation activity.
  • a library of cyclotides each inserted with a random peptide is screened to identify individual members of the library that exhibit a desired biological activity such as membrane translocation activity, and in particular transfer across the BBB when delivered to an animal.
  • transfer across the blood-brain barrier or “crossing the blood-brain barrier” or other variations thereof it is meant that the polypeptide is delivered to an animal and is capable of passing into the brain of the animal by traversing blood vessel walls in the brain.
  • a method for screening for BBB transfer includes the steps of: a) constructing a cyclotide sequence display library in which random peptides are inserted into the cyclotide scaffold (e.g. into a loop therein, e.g. loop 1); b) expressing the sequence library in order to obtain expressed cyclotides, or polypeptides that comprise the cyclotides; c) administering the expressed cyclotides or polypeptides that comprise the cyclotides to an animal; d) recovering any cyclotide from the brain of the mammal; e) determining the sequence of the recovered cyclotide or peptide contained thereins.
  • the animal used in such a screen is typically a bird or mammal, and may be selected from humans, primates, cattle, sheep, rodents, cats, dogs, and rabbits.
  • the library of cyclotides may be suitably administered by oral gavage or inclusion within normal animal feed. Recovery of the cyclotides from the body of the animal may be via biopsy, or in the case of non-human animals via sacrifice of the animal and histological and pathological analysis of the tissues in the body. In this way it is also possible to identify members of the library of cyclotides that exhibit tissue specificity and/or the availability to cross various additional barriers within the body of the animal.
  • modified cyclotides that are found in a brain biopsy of a screened non-human animal would be considered as demonstrating the ability of being able to cross the BBB.
  • the cyclotides of the invention are comprised within a phage display library and the determination of BBB transfer is made by analysing which cyclotides are capable of facilitating transport of an associated phage particle as a whole into the brain of an animal which has been fed or intravenously injected with at least a part of the phage display library.
  • the further analysis involves the isolation of cyclotides by infection of bacteria as an amplification step, isolating the phage or phagemid DNA, and cloning the DNA sequence encoding the candidate cyclotides contained in said phage or phagemid DNA into a suitable expression vector.
  • an infection step can also allow the amplification of the cyclotides.
  • cyclotides can be amplified at this stage by other appropriate methods, for example by PCR of the nucleic acids encoding said cyclotides or the transformation of said nucleic acid into an appropriate host cell (in the context of a suitable expression vector).
  • the DNA encoding the cyclotides are cloned in a suitable expression vector, the DNA encoding the cyclotides can be sequenced or the protein can be expressed in a soluble form, e.g., including according to the methods provided herein, and subjected to appropriate binding studies to further characterize the candidates at the protein level.
  • the peptide contained in a selected cyclotide can be identified and can be assessed for binding to the BBB-R.
  • binding studies will depend on the nature of the binders, and include, but are not limited to ELISA, filter screening assays, FACS, or immunofluorescence assays, BiaCore affinity measurements or other methods to quantify binding constants, staining tissue slides or cells and other immunohistochemistry methods.
  • ELISA ELISA
  • filter screening assays FACS
  • immunofluorescence assays BiaCore affinity measurements or other methods to quantify binding constants, staining tissue slides or cells and other immunohistochemistry methods.
  • BiaCore affinity measurements BiaCore affinity measurements or other methods to quantify binding constants, staining tissue slides or cells and other immunohistochemistry methods.
  • identified peptides can be modified by histidine or alanine scanning to identify peptides with altered binding or affinity. For instance, a library of mutant peptides can be generated and binding of each variant for the BBB-R target molecule can be assessed. Any binding assay known to a skilled artisan can be used, including any as described above.
  • histidine scanning is carried out to mutate or vary amino acid residues in an identified peptide to histidine.
  • histidine scanning can be used to identify pH-sensitive antigen binding, for example to identify peptides that have reduced binding at acidic pH compared to neutral pH.
  • a receptor-binding peptide bind to a BBB-R at neutral pH (e.g. pH 7.0-7.4) but not bind or have reduced binding at pH 5.0 such as present in an endosome so that the binding molecule containing the peptide (e.g. modified cyclotide) is released into the blood-brain barrier and not recycled.
  • alanine scanning is carried out to mutate or vary amino acid residues in an identified peptide to alanine. In some embodiments, alanine scanning is useful to identify residues that can be mutated with retention of binding function.
  • peptide that binds to a BBB-R by amino acid sequence, including from a sequence library.
  • the peptide is 6 to 50 amino acids in length and comprises the sequence set forth in any one of SEQ ID NOS: 26-34 or 49-54.
  • the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids and comprises the sequence set forth in any one of SEQ ID NOS: 26-34 or 49-54.
  • the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO: 26.
  • the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO: 49.
  • the peptide is set forth in any one of SEQ ID NOS: 26-34 or 49-54. In some embodiments, the peptide is set forth in SEQ ID NO:26. In some embodiments, the peptide is set forth in SEQ ID NO: 49. In some embodiments, the peptide moiety binds the transferrin receptor (TrfR). In some embodiments, the TrfR is mouse. In some embodiments, the TrfR is human. In some embodiments, the peptide has blood-brain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises a sequence that has at least 85%, at least 90%, at least 95% amino acid sequence identity to the sequence set forth in any one of SEQ ID NOS: 26-34 or 49-54. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 26- 34 or 49-54. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 26-34 or 49-54.
  • the amino acid substitution(s) is substitution of an amino acid residue to an alanine. In some embodiments, the amino acid substitution(s) is substitution of an amino acid residue to histidine. In some embodiments, the peptide moiety binds the transferrin receptor (TrfR). In some embodiments, the TrfR is mouse. In some embodiments, the TrfR is human. In some embodiments, the peptide has blood-brain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises a sequence that has at least 85%, at least 90%, at least 95% amino acid sequence identity to the sequence set forth in any one of SEQ ID NO: 26. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 26. In some embodiments, the amino acid substitution(s) is substitution of an amino acid residue to an alanine. In some embodiments, the amino acid substitution(s) is substitution of an amino acid residue to histidine.
  • the peptide is 6 to 50 amino acids in length and comprises the sequence set forth in any one of SEQ ID NOS: 55 or 117-128. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids and comprises the sequence set forth in any one of SEQ ID NOS: 55 or 117-128. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO: 55.
  • the peptide is set forth in any one of SEQ ID NOS: 55 or 117-128. In some embodiments, the peptide is set forth in SEQ ID NO:55. In some embodiments, the peptide moiety binds the transferrin receptor (TrfR). In some embodiments, the TrfR is mouse. In some embodiments, the TrfR is human. In some embodiment, the peptide has blood-brain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises the sequence set forth in any one of SEQ ID NOS: 10-23. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids and comprises the sequence set forth in any one of SEQ ID NOS: 10-23. In some embodiments, the peptide is set forth in any one of SEQ ID NOS: 10-23. In some embodiments, the peptide moiety binds the leptin receptor (ObR). In some embodiments, the ObR is human. In some embodiment, the peptide has blood-brain barrier translocation activity.
  • ObR leptin receptor
  • the peptide is 6 to 50 amino acids in length and comprises a sequence that has at least 85%, at least 90%, at least 95% amino acid sequence identity to the sequence set forth in any one of SEQ ID NOS: 10-23. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 10-23. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 210-23.
  • the amino acid substitution(s) is substitution of an amino acid residue to an alanine. In some embodiments, the amino acid substitution(s) is substitution of an amino acid residue to histidine. In some embodiments, the peptide moiety binds the leptin receptor (ObR). In some embodiments, the ObR is human. In some embodiments, the peptide has blood-brain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises the sequence set forth in any one of SEQ ID NOS: 24 or 25. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids and comprises the sequence set forth in any one of SEQ ID NOS: 24 or 25. In some embodiments, the peptide is set forth in SEQ ID NO: 24.
  • the peptide is set forth in SEQ ID NO: 25. In some embodiments, the peptide moiety binds ErbB3. In some embodiments, the ErBB3 is human. In some embodiment, the peptide has blood-brain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises a sequence that has at least 85%, at least 90%, at least 95% amino acid sequence identity to the sequence set forth in any one of SEQ ID NOS: 24 or 25. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 24 or 25. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 24 or 25.
  • the amino acid substitution(s) is substitution of an amino acid residue to an alanine. In some embodiments, the amino acid substitution(s) is substitution of an amino acid residue to histidine. In some embodiments, the peptide moiety binds the ErbB3. In some embodiments, the ErbB3 is human. In some embodiments, the peptide has blood-brain barrier translocation activity. [OHl] In some embodiments, the peptide is 6 to 50 amino acids in length and comprises the sequence set forth in any one of SEQ ID NOS: 35 or 36.
  • the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids and comprises the sequence set forth in any one of SEQ ID NOS: 35 or 36.
  • the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO:35.
  • the peptide is set forth in SEQ ID NO: 35.
  • the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO:36. In some embodiments, the peptide is set forth in any one of SEQ ID NO: 36. In some embodiments, the peptide moiety binds IGFR. In some embodiments, the IGFR is human. In some embodiment, the peptide has blood-brain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises a sequence that has at least 85%, at least 90%, at least 95% amino acid sequence identity to the sequence set forth in any one of SEQ ID NOS: 35 or 36. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 35 or 36. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 35 or 36.
  • the amino acid substitution(s) is substitution of an amino acid residue to an alanine. In some embodiments, the amino acid substitution(s) is substitution of an amino acid residue to histidine. In some embodiments, the peptide moiety binds the IGFR. In some embodiments, the IGFR is human. In some embodiments, the peptide has blood-brain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises the sequence set forth in any one of SEQ ID NOS: 37 or 38. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids and comprises the sequence set forth in any one of SEQ ID NOS: 37 or 38. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO:37.
  • the peptide is set forth in SEQ ID NO: 37. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO:38. In some embodiments, the peptide is set forth in SEQ ID NO: 38. In some embodiments, the peptide moiety binds RAGE. In some embodiments, the RAGE is human. In some embodiment, the peptide has blood-brain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises a sequence that has at least 85%, at least 90%, at least 95% amino acid sequence identity to the sequence set forth in any one of SEQ ID NOS: 37 or 38. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 37 or 38. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 37 or 38.
  • the amino acid substitution(s) is substitution of an amino acid residue to an alanine. In some embodiments, the amino acid substitution(s) is substitution of an amino acid residue to histidine. In some embodiments, the peptide moiety binds the RAGE. In some embodiments, the RAGE is human. In some embodiment, the peptide has blood-brain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises the sequence set forth in any one of SEQ ID NOS: 39-48. In some embodiments, the peptide is 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 10 to 30 amino acids, 10 to 24 amino acids, 10 to 18 amino acids, 10 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids and comprises the sequence set forth in any one of SEQ ID NOS: 39-48. In some embodiments, the peptide is set forth in any one of SEQ ID NOs: 39-48.
  • the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO:39. In some embodiments, the peptide is set forth in SEQ ID NO: 39. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO:41. In some embodiments, the peptide is set forth in SEQ ID NO: 41. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises the sequence set forth in SEQ ID NO:43. In some embodiments, the peptide is set forth in SEQ ID NO: 43. In some embodiments, the peptide moiety binds LRP- 1. In some embodiments, the LRP-1 is human. In some embodiment, the peptide has bloodbrain barrier translocation activity.
  • the peptide is 6 to 50 amino acids in length and comprises a sequence that has at least 85%, at least 90%, at least 95% amino acid sequence identity to the sequence set forth in any one of SEQ ID NOS: 39-48. In some embodiments, the peptide is 6 to 50 amino acids in length and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NOS: 39-48. In some embodiments, the peptide is 12 to 18 amino acids in length, and comprises a sequence that has 1, 2 , 3 or 4 amino acid substitutions compared to the sequence set forth in any one of SEQ ID NO: 39, 41 or 43.
  • the amino acid substitution(s) is substitution of an amino acid residue to an alanine. In some embodiments, the amino acid substitution(s) is substitution of an amino acid residue to histidine. In some embodiments, the peptide moiety binds the LRP-1. In some embodiments, the LRP-1 is human. In some embodiment, the peptide has blood-brain barrier translocation activity.
  • the peptide has blood-brain barrier translocation activity. In some of any of the preceding embodiments, the peptide binds to a receptor involved in blood-brain barrier transcytosis.
  • Binding Molecules e.g. Cyclotide Scaffolds and Modified Cyclotides
  • binding molecules that comprise a peptide that binds to a BBB-R, including any set forth in Section I. A. above.
  • the binding molecule provides a stable scaffold (also interchangeably used with the term “backbone”) into which the peptide can be inserted.
  • the binding molecule is a cysteine-knot protein.
  • the binding molecule is a cyclotide.
  • variable loops of antibodies have been extensively engineered to produce peptides having improved binding (e.g. affinity and/or specificity) to known ligands, and also to expand the binding substrates for particular antibody frameworks (see for example, Knappik et al., (2000) J. Mol. Biol., 296, 57-86; and EP 1025218).
  • the engineering of non-antibody frameworks has been reviewed, for example, by Hosse et al., (2006), Protein Sci., 15, 14-27.
  • Suitable scaffolds may include modified whey acidic protein (WAP) domain containing polypeptides, such as those described in International patent application published as WO-A-2012073045. It will be appreciated by the skilled person that suitable scaffolds are not limited solely to the WAP domain of human elafin (trappin-2) but may include other human trappins (e.g. SLPI) or non-human trappins (e.g. from porcine, bovine or simian sources). In addition, the provided embodiments extends to cyclotides comprised within other non-trappin members of the WAP domain family. Further, provided embodiments also relate to employing plant cyclotides as scaffolds.
  • WAP modified whey acidic protein
  • the molecules provided herein that employ cysteine-knot proteins, such as cyclotides, as scaffolds for insertion of the peptides may be used as carriers for delivery of biological agents, such as protein therapeutics, across the blood brain barrier.
  • the cyclotide is a linear or cyclic amino acid sequence containing a structure referred to herein as a "cysteine knot".
  • a cysteine knot occurs when a disulfide bond passes through a closed cyclic loop formed by two other disulfide bonds and the amino acids in the backbone.
  • reference herein to a "cysteine knot" includes reference to structural equivalents thereof which provide similar constraints to the three-dimensional structure of the cyclic backbone.
  • cysteine knot backbone may also be achieved by engineering suitable covalent bonds or other forms of molecular associations. All such modifications to the cyclic backbone which result in retention of the three-dimensional knotted topology conferred by the cysteine knot are encompassed by the provided embodiments.
  • a cysteine knot is characterized by a knot formed by three disulfide bonds
  • the provided embodiments extends to molecules comprising only two disulfide bonds.
  • the molecular framework may need to be further stabilized using other means or the molecular framework may retain suitable activity despite a change in three-dimensional structure caused by the absence of a third disulfide bond.
  • the cysteine knot backbone may comprise more than three disulfide bonds such as occurring in a double or multiple cysteine knot arrangement or in a single cysteine knot arrangement supplement by one or two additional disulfide bonds.
  • Cysteine-knot microproteins include a naturally occurring family of cysteine-knot microproteins or cyclotides found in various plant species.
  • Cysteine- knot microproteins are small peptides, typically consisting of about 30-40 amino acids, which can be found naturally as cyclic or linear forms, where the cyclic form has no free N- or C-terminal amino or carboxyl end. They have a defined structure based on three intra-molecular disulfide bonds and a small triple stranded P-sheet (Craik et al., 2001; Toxicon 39, 43-60).
  • the cyclic proteins exhibit conserved cysteine residues defining a structure referred to herein as a "cysteine knot".
  • This family includes both naturally occurring cyclic molecules and their linear derivatives as well as linear molecules which have undergone cyclization. These molecules are useful as molecular framework structures having enhanced stability over less structured peptides. (Colgrave and Craik, 2004; Biochemistry 43, 5965-5975). However, these molecules are not themselves capable of crossing the bloodbrain barrier to any great extent, and cannot be used as neuro-therapeutic agents themselves or act as carriers of therapeutic peptide or protein agents.
  • the main cyclotide features are a remarkable stability due to the cysteine knot, a small size making them readily accessible to chemical synthesis, and an excellent tolerance to sequence variations.
  • the provided embodiments are based on the recognition herein that cyclotides therefore appear as appealing leads or scaffolds for peptide drug design.
  • the cyclotide scaffold is found in almost 30 different protein families among which conotoxins, spider toxins, squash inhibitors, agouti -related proteins and plant cyclotides are the most populated families. Cyclotides from plants in the Rubiaceae and Violaceae families are for the most part found to be head-to-tail cyclic peptides (Craik et al. 2010. Cell. Mol. Life Sci.
  • cyclotides are commonly found in plants.
  • cyclotides of the invention are derived from linear or cyclic form of cyclotides of the Momordicae, Rubiaceae and Violaceae, plant species.
  • cyclotides of the invention are derived from linear or cyclic form of cyclotides of the Momordicae species including the squash serine protease inhibitor family (Otlewski & Korowarsch Acta Biochim Pol. 1996;43(3):431-44) and in a more preferred aspect from Momordica cochinchinensis trypsin inhibitors.
  • the Momordica cochinschinenis trypsin inhibitor include inhibitorsMCoTI-I (SEQ ID NO: 1) and -II (SEQ ID NO: 2) (naturally cyclic) and MCoTI-III (naturally linear) (SEQ ID NO: 3) below.
  • Mcoti-I GGVCPKILQRCRRDSDSPGACICRGNGYCGSGSD SEQ ID NO: 1
  • Mcoti-II GGVCPKILKKCRRDSDSPGACICRGNGYCGSGSD SEQ ID NO: 2
  • Mcoti-III ERACPRILKKCRRDSDSPGACICRGNGYCG SEQ ID NO : 3
  • the peptide is any as described in Section I. A.
  • the peptide is inserted into a loop of the unmodified or wild-type cyclotide.
  • a modified cyclotide sequence provided herein may be defined as having a cysteine knot backbone moiety and a peptide that is a blood-brain barrier translocation moiety, said modified cyclotide comprising: i) a peptide that binds a BBB-R, wherein said peptide is about 2 to 50 amino acid residues length; and ii) a cysteine knot backbone grafted to said peptide of clause i), wherein said cysteine knot backbone comprises the structure (I):
  • Ci to Ce are cysteine residues; wherein each of Ci and C 4 , C 2 and Cs, and C 3 and Ce are connected by a disulfide bond to form a cysteine knot; wherein each X represents an amino acid residue in a loop, wherein said amino acid residues are the same or different; wherein d is about 1-2; wherein one or more of loops 1, 2, 3, 5 or 6 have an amino acid sequence comprising the sequence of said peptide of clause i), wherein any loop comprising said sequence of said peptide of clause i) comprises 2 to about 50 amino acids, and wherein for any of loops 1, 2, 3, 5, or 6 that do not contain said sequence of said peptide of clause i), a, b, c, e, and f, are the same or different, and are each any number from 3-10, and b, c, e, and f are each any number from 1 to 20.
  • the peptide is
  • the unmodified or wildtype cyclotide can be a cyclotide set forth in any one of SEQ ID NOS: 1-3 to which one or more loops thereof is inserted or substituted by one or more amino acid sequences, e.g. a peptide of 2 to 50 amino acids, such as any described in Section I. A.
  • the unmodified or wildtype cyclotide can be a cyclotide set forth in any one of SEQ ID NO: 1 to which one or more loops thereof is inserted or substituted by one or more amino acid sequences, e.g. a peptide of 2 to 50 amino acids, such as any described in Section I. A.
  • the unmodified or wildtype cyclotide can be a cyclotide set forth in SEQ ID NO:2 to which one or more loops thereof is inserted or substituted by one or more amino acid sequences, e.g. a peptide of 2 to 50 amino acids, such as any described in Section I. A.
  • the unmodified or wildtype cyclotide can be a cyclotide set forth in any one of SEQ ID NO:3 to which one or more loops thereof is inserted or substituted by one or more amino acid sequences, e.g. a peptide of 2 to 50 amino acids, such as any described in Section I. A.
  • the provided modified cyclotides of the invention exhibit improved or enhanced binding to a receptor involved in transcytosis across the blood-brain barrier, such as ta BBB-R, e.g. the human transferrin receptor.
  • the provided modified cyclotides possess enhanced blood-brain barrier translocation characteristics.
  • the improvement or enhancement is as compared to the unmodified cyclotide from which the modified cyclotide is derived, such as a wild-type or natural cyclotide, including a cyclotide set forth in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
  • the provided cyclotides contain a sequence of amino acids or analogues thereof forming a cysteine-knot backbone wherein said cysteine-knot backbone comprises sufficient disulfide bonds or chemical equivalents thereof, to confer a knotted topology on the three- dimensional structure of said cysteine-knot backbone and wherein at least one exposed amino acid residue such as on one or more beta turns and/or within one or more loops, is inserted or substituted relative to the naturally occurring cyclotide amino acid sequence.
  • the inserted or substituted loop is loop 1 of a native or naturally occurring cyclotide sequence.
  • the inserted or substituted loop is loop 2 of a native or naturally occurring cyclotide sequence.
  • the inserted or substituted loop is loop 3 of a native or naturally occurring cyclotide sequence. In some embodiments, the inserted or substituted loop is loop 5 of a native or naturally occurring cyclotide sequence. In some embodiments, the inserted or substituted loop is loop 6 of a native or naturally occurring cyclotide sequence. In some embodiments, the provided cyclotides exhibit enhanced translocation behaviour compared with the unmodified or native parental cyclotide.
  • the provided modified cyclotides are derived from loop replacement libraries based on Mcoti-I (SEQ ID NO: 1).
  • a modified cyclotide comprising a blood-brain barrier translocation peptide moiety that is inserted into or that substitutes or replaces one or more amino acids of a loop of the cyclotide Mcoti-I (SEQ ID NO: 1).
  • the peptide has an amino acid sequence that is about 2 to 50 amino acid residues, such as any described in Section I. A.
  • the loop into which the peptide is inserted or subtituted is loop 1.
  • the modified cyclotide has the sequence of SEQ ID NO:1 in which residues in loop 1 between the first two cysteine are replaced by the binding peptide.
  • the peptide is inserted between cysteine 4 (Cys4) and cysteine 11 (Cysl 1) of SEQ ID NO: 1.
  • the loop into which the peptide is inserted or substituted is loop 5.
  • the modified cyclotide has the sequence of SEQ ID NO: 1 in which residues in loop 5 between the last two cysteine are replaced by the binding peptide.
  • the peptide is inserted between cysteine 23 (Cys23) and cysteine 29 (Cys29) of SEQ ID NO: 1.
  • the loop into which the peptide is inserted or substituted is loop 6, such as formed subject to cyclization.
  • the modified cyclotide has the sequence of SEQ ID NO: 1 in which residues in loop 6 between the first and last cysteine (after cyclization) are replaced by the binding peptide.
  • the peptide is inserted between cysteine 29 (Cys29) and cysteine 4 (Cys4) of SEQ ID NO: 1.
  • the provided modified cyclotides are derived from loop replacement libraries based on Mcoti-II (SEQ ID NO: 2).
  • a modified cyclotide comprising a blood-brain barrier translocation peptide moiety that is inserted into or that substitutes or replaces one or more amino acids of a loop of the cyclotide Mcoti-II (SEQ ID NO:2).
  • the peptide has an amino acid sequence that is about 2 to 50 amino acid residues, such as any described in Section I. A.
  • the loop into which the peptide is inserted or subtituted is loop 1.
  • the modified cyclotide has the sequence of SEQ ID NO:2 in which residues in loop 1 between the first two cysteine are replaced by the binding peptide.
  • the peptide is inserted between cysteine 4 (Cys4) and cysteine 11 (Cysl 1) of SEQ ID NO:2.
  • the loop into which the peptide is inserted or substituted is loop 5.
  • the modified cyclotide has the sequence of SEQ ID NO:2 in which residues in loop 5 between the last two cysteine are replaced by the binding peptide.
  • the peptide is inserted between cysteine 23 (Cys23) and cysteine 29 (Cys29) of SEQ ID NO:2.
  • the loop into which the peptide is inserted or substituted is loop 6, such as formed subject to cyclization.
  • the modified cyclotide has the sequence of SEQ ID NO:2 in which residues in loop 6 between the first and last cysteine (after cyclization) are replaced by the binding peptide.
  • the peptide is inserted between cysteine 29 (Cys29) and cysteine 4 (Cys4) of SEQ ID NO:2.
  • the provided modified cyclotides are derived from loop replacement libraries based on Mcoti-III (SEQ ID NO: 3).
  • a modified cyclotide comprising a blood-brain barrier translocation peptide moiety that is inserted into or that substitutes or replaces one or more amino acids of a loop of the cyclotide Mcoti-III (SEQ ID NO:3).
  • the peptide has an amino acid sequence that is about 2 to 50 amino acid residues, such as any described in Section I. A.
  • the loop into which the peptide is inserted or subtituted is loop 1.
  • the modified cyclotide has the sequence of SEQ ID NO: 3 in which residues in loop 1 between the first two cysteine are replaced by the binding peptide.
  • the peptide is inserted between cysteine 4 (Cys4) and cysteine 11 (Cysl 1) of SEQ ID NO:3.
  • the loop into which the peptide is inserted or substituted is loop 5.
  • the modified cyclotide has the sequence of SEQ ID NO:3 in which residues in loop 5 between the last two cysteine are replaced by the binding peptide.
  • the peptide is inserted between cysteine 23 (Cys23) and cysteine 29 (Cys29) of SEQ ID NO:3.
  • the loop into which the peptide is inserted or substituted is loop 6, such as formed subject to cyclization.
  • the modified cyclotide has the sequence of SEQ ID NO:3 in which residues in loop 6 between the first and last cysteine (after cyclization) are replaced by the binding peptide.
  • the peptide is inserted between cysteine 29 (Cys29) and cysteine 4 (Cys4) of SEQ ID NO:3.
  • the peptide that is inserted or replaced into an unmodified cyclotide e.g. the cyclotide Mcoti-I (SEQ ID NO: 1)
  • the peptide that is inserted or replaced into an unmodified cyclotide e.g. the cyclotide Mcoti-II (SEQ ID NO:2)
  • the peptide that is inserted or replaced into an unmodified cyclotide e.g. the cyclotide Mcoti-III (SEQ ID NO:3)
  • the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 aminio acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.
  • the peptide is 2 to 30 amino acids, such as 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids.
  • the peptide is 14 to 20 amino acids.
  • the peptide is 10 amino acids.
  • the peptide is 11 amino acids.
  • the peptide is 12 amino acids.
  • the peptide is 13 amino acids.
  • the peptide is 14 amino acids.
  • the peptide is 15 amino acids. In some embodiments, the peptide is 16 amino acids. In some embodiments, the peptide is 17 amino acids. In some embodiments, the peptide is 18 amino acids. In some embodiments, the peptide is 19 amino acids. In some embodiments, the peptide is 20 amino acids.
  • a provided modified cyclotide has an amino acid sequence that exhibits at least 85% sequence identity to any of SEQ ID NOS: 56-101 or 105-116, wherein the modified cyclotide has blood-brain barrier translocation activity and/or binds to a receptor involved in blood-brain barrier transcytosis. In some embodiments, a provided modified cyclotide has an amino acid sequence that exhibits at least 90% sequence identity to any of SEQ ID NOS: 56-101 or 105-116, wherein the modified cyclotide has blood-brain barrier translocation activity and/or binds to a receptor involved in blood-brain barrier transcytosis.
  • a provided modified cyclotide has an amino acid sequence that exhibits at least 95% sequence identity to any of SEQ ID NOS: 56-101 or 1 OS- 116, wherein the modified cyclotide has blood-brain barrier translocation activity and/or binds to a receptor involved in blood-brain barrier transcytosis.
  • the sequence identity it at or about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to any of SEQ ID NOS: 56-101 or 105-116.
  • the modified cyclotide binds to a receptor involved in blood-brain barrier transcytosis, such as a BBB-R.
  • the modified cyclotide has blood-brain barrier translocation activity, such as measured following subcutaneous administration to a subject.
  • the modified cyclotide binds to a receptor involved in blood-brain barrier transcytosis, i.e. BBB-R, and has blood-brain barrier translocation activity when administered subcutaneously to a subject.
  • the modified cyclotide binds to a BBB-R that is expressed on brain endothelial cells.
  • the BBB-R is selected from the transferrin receptor (TrfR); the lactoferrin receptor (LtfR); the leptin receptor (LEP-R, or also known as OB-R); the receptor tyrosine-protein kinase (ErbB3), a insulin receptor, such as Insulin Receptor A (IRA), IGF-1 Receptor (IGF-1R), IGF-II Receptor (IGF-IIR), RXFP1, RXFP2, RXFP3 and RXFP4; low-density lipoprotein receptor- related protein 1 (LRP-1), the receptor for advanced glycation end products (RAGE); heparin binding EGF like growth factor receptor (HB EGFR); the intercellular adhesion molecule 1 (ICAM1), intercellular adhesion molecule 2
  • IGF-1 insulin receptor
  • IGF-1 insulin receptor
  • the modified cyclotides are derived from loop replacement libraries based on Mcoti-II (SEQ ID NO: 2) and bind to the transferrin receptor (e.g. human transferrin receptor).
  • the loop replacement is replacement of at least one loop, such as loop 1, with a peptide that comprises the sequence set forth in any one of SEQ ID NOS: 26-34 and 49-54.
  • the peptide is the peptide set forth in any one of SEQ ID NOS: 26-34 and 49-54.
  • the peptide is the peptide set forth in SEQ ID NO:26.
  • the peptide is the peptide set forth in SEQ ID NO: 49.
  • the modified cyclotide comprises the sequence set forth in any one of SEQ ID NOS: 72-80 and 95-100. In some embodiments, the modified cyclotide is set forth in any one of SEQ ID NOS: 72-80 and 95-100. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:72. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 95. In some embodiments, the peptide contained in the modified cyclotide has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in any one of SEQ ID NOS: 26-34 and 49-54. In some embodiments, the amino acid substitution(s) is to a histidine.
  • the amino acid substitution(s) is to an alanine.
  • the modified cyclotide comprises the peptide sequence set forth in any one of SEQ ID NOS: 55 and 117-128. In some embodiments, the peptide sequenceisset forth in any one of SEQ ID NOS: 55 and 117-128. In some embodiments, the modified cyclotide comprises the sequence set forth in any one of SEQ ID NOS: 101 and 105-116. In some embodiments, the modified cyclotide is set forth in any one of SEQ ID NOS: 101 and 105- 116. In some embodiments, the peptide sequence is set forth in SEQ ID NO:55 and the modified cyclotide is set forth in SEQ ID NO: 101.
  • the modified cyclotide is a modified cyclotide that binds to the transferrin but has altered binding to transferrin receptor compared to the cyclotide set forth in any one of SEQ ID NOS: 72-80 and 95-100. In some embodiments, the modified cyclotide has altered binding to transferrin receptor compared to the cyclotide set forth in SEQ ID NOS: 72.
  • the modified cyclotide is a modified cyclotide that has reduced binding affinity to the transferrin receptor compared to the modified cyclotide set forth in SEQ ID NO:72, such as reduced by 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10.0-fold or more.
  • the modified cyclotide has 1, 2, 3, 4 or 5 amino acid differences compared to the cyclotide set forth in SEQ ID NO:72.
  • the modified cyclotide has one or more amino acid substitutions compared to the cyclotide set forth in SEQ ID NO:72, wherein the one or more amino acid substitutions are selected from the group consisting of W9H, LI 1H, S13H, W14H and G15H.
  • the peptide is a peptide set forth in any one of SEQ ID NOS: 55, 122, 124, 126 or 127.
  • the peptide is set forth in SEQ ID NO: 55.
  • the modified cyclotide is modified by loop replacement (e.g.
  • the modified cyclotide comprises the sequence set froth in SEQ ID NO: 110.
  • the modified cyclotide is modified by loop replacement (e.g. loop 1) of Mcoti-II (SEQ ID NO: 2) with the peptide set forth in SEQ ID NO: 124, for example the modified cyclotide comprises the sequence set forth in SEQ ID NO: 112.
  • the modified cyclotide is modified by loop replacement (e.g.
  • the modified cyclotide comprises the sequence set forth in SEQ ID NO: 114.
  • the modified cyclotide is modified by loop replacement (e.g. loop 1) of Mcoti-II (SEQ ID NO: 2) with the peptide set forth in SEQ ID NO: 127, for example the modified cyclotide comprises the sequence set forth in SEQ ID NO:115.
  • the modified cyclotide is modified by loop replacement (e.g.
  • the modified cyclotide comprises the sequence set forth in SEQ ID NO: 101.
  • the binding affinity to the transferrin receptor is pH- dependent, such that the modified cyclotide exhibits greater binding affinity to the transferrin receptor at neutral pH (e.g. 7.0-7.4) than acidic pH of the endosome (e.g. pH of 4.5 to 6.5, such as at or about pH 5.0).
  • such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics. Without wishing to be bound by theory, in some aspects a modified cyclotide with reduced binding to the transferrin receptor is released into the blood-brain barrier and not recycled back.
  • the modified cyclotides are derived from loop replacement libraries based on Mcoti-II (SEQ ID NO: 2) and bind to the leptin receptor (e.g. human leptin receptor, also called Hu ObR).
  • the loop replacement is replacement of at least one loop, such as loop 1, with a peptide that comprises the sequence set forth in any one of SEQ ID NOS: 10-23.
  • the peptide is the peptide set forth in any one of SEQ ID NOS: 10-23.
  • the modified cyclotide comprises the sequence set forth in any one of SEQ ID NOS: 56-69.
  • the modified cyclotide is set forth in any one of SEQ ID NOS: 56-69.
  • the modified cyclotide is a modified cyclotide that binds to the leptin receptor but has altered binding to the leptin receptor compared to the cyclotide set forth in any one of SEQ ID NOS: 56-69.
  • the binding affinity to the leptin receptor is reduced, such as reduced by 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fole, 10.0-fold or more.
  • the binding affinity to the leptin receptor is pH- dependent, such that the modified cyclotide exhibits greater binding affinity to the leptin receptor at neutral pH (e.g. 7.0-7.4) than acidic pH of the endosome (e.g.
  • the modified cyclotide has a loop with a peptide that has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in any one of SEQ ID NOS: 10-23.
  • the modified cyclotide is modified by loop replacement (e.g. loop 1) of Mcoti-II (SEQ ID NO: 2) with a peptide that has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in any one of SEQ ID NOS: 10-23.
  • the amino acid substitution(s) is to a histidine.
  • the amino acid substitution(s) is to an alanine.
  • such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.
  • the modified cyclotides are derived from loop replacement libraries based on Mcoti-II (SEQ ID NO: 2) and bind to ErbB3 (e.g. human ErbB3).
  • the loop replacement is replacement of at least one loop, such as loop 1, with a peptide that comprises the sequence set forth in SEQ ID NO: 24 or 25.
  • the peptide is the peptide set forth in SEQ ID NO: 24 or 25.
  • the modified cyclotide comprises the sequence set forth in SEQ ID NO: 70 or 71.
  • the modified cyclotide is set forth in SEQ ID NOS: 70 or 71.
  • the modified cyclotide is a modified cyclotide that binds to ErbB3 but has altered binding to ErB3 compared to the cyclotide set forth in SEQ ID NOS: 70 or 71.
  • the binding affinity to the ErbB3 is reduced, such as reduced by 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fole, 10.0-fold or more.
  • the binding affinity to ErbB3 is pH-dependent, such that the modified cyclotide exhibits greater binding affinity to ErB3 at neutral pH (e.g. 7.0-7.4) than acidic pH of the endosome (e.g.
  • the modified cyclotide has a loop with a peptide that has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in SEQ ID NO: 24 or 25.
  • the modified cyclotide is modified by loop replacement (e.g. loop 1) of Mcoti-II (SEQ ID NO: 2) with a peptide that has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in any one of SEQ ID NOS: 24 or 25.
  • the amino acid substitution(s) is to a histidine.
  • the amino acid substitution(s) is to an alanine.
  • such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.
  • the modified cyclotides are derived from loop replacement libraries based on Mcoti-II (SEQ ID NO: 2) and bind to the IGFR (e.g. human IGFR).
  • the loop replacement is replacement of at least one loop, such as loop 1, with a peptide that comprises the sequence set forth in SEQ ID NO: 35 or 36.
  • the peptide is the peptide set forth in SEQ ID NOS: 35 or 36.
  • the peptide is the peptide set forth in SEQ ID NO:36.
  • the modified cyclotide comprises the sequence set forth in SEQ ID NO: 81 or 82.
  • the modified cyclotide is set forth in SEQ ID NO: 81 or 82. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 81. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 82.
  • the modified cyclotide is a modified cyclotide that binds to IGFR but has altered binding to IGFR compared to the cyclotide set forth in SEQ ID NO: 81 or 82. In some embodiments, the modified cyclotide has altered binding to IGFR compared to the cyclotide set forth in SEQ ID NOS: 82. In some embodiments, the binding affinity to the IGFR is reduced, such as reduced by 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10.0-fold or more.
  • the binding affinity to IGFR is pH-dependent, such that the modified cyclotide exhibits greater binding affinity to IGFR at neutral pH (e.g. 7.0-7.4) than acidic pH of the endosome (e.g. pH of 4.5 to 6.5, such as at or about pH 5.0).
  • the modified cyclotide has a loop with a peptide that has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in SEQ ID NO: 35 or 36.
  • the modified cyclotide is modified by loop replacement (e.g.
  • the amino acid substitution(s) is to a histidine. In some embodiments, the amino acid substitution(s) is to an alanine. In some embodiments, such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.
  • the modified cyclotides are derived from loop replacement libraries based on Mcoti-II (SEQ ID NO: 2) and bind to RAGE (e.g. human RAGE).
  • the loop replacement is replacement of at least one loop, such as loop 1, with a peptide that comprises the sequence set forth in SEQ ID NO: 37 or 38.
  • the peptide is the peptide set forth in SEQ ID NO: 37 or 38.
  • the modified cyclotide comprises the sequence set forth in SEQ ID NO: 83 or 84.
  • the modified cyclotide is set forth in SEQ ID NOS: 83 or 84.
  • the modified cyclotide is set forth in SEQ ID NO: 83.
  • the modified cyclotide is set forth in SEQ ID NO:84.
  • the modified cyclotide is a modified cyclotie that bnds to RAGE but has altered binding to RAGE compared to the cyclotide set forth in SEQ ID NOS: 83 or 84.
  • the binding affinity to the RAGE is reduced, such as reduced by 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10.0-fold or more.
  • the binding affinity to RAGE is pH-dependent, such that the modified cyclotide exhibits greater binding affinity to ErB3 at neutral pH (e.g. 7.0-7.4) than acidic pH of the endosome (e.g. pH of 4.5 to 6.5, such as at or about pH 5.0).
  • the modified cyclotide has a loop with a peptide that has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in SEQ ID NO:37 or 38.
  • the modified cyclotide is modified by loop replacement (e.g. loop 1) of Mcoti-II (SEQ ID NO: 2) with a peptide that has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in any one of SEQ ID NOS: 37 or 38.
  • the amino acid substitution(s) is to a histidine.
  • the amino acid substitution(s) is to an alanine.
  • such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.
  • the modified cyclotides are derived from loop replacement libraries based on Mcoti-II (SEQ ID NO: 2) and bind to the LRP-1 (e.g. human LRP-1).
  • the loop replacement is replacement of at least one loop, such as loop 1, with a peptide that comprises the sequence set forth in any one of SEQ ID NOS: 39-48.
  • the peptide is the peptide set forth in any one of SEQ ID NOS: 39-48.
  • the modified cyclotide comprises the sequence set forth in any one of SEQ ID NOS: 85-94.
  • the modified cyclotide is set forth in any one of SEQ ID NOS: 85-94. In some embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO: 87. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 87. In some embodiments, the modified cyclotide comprises the sequence set forth in SEQ ID NO: 89. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 89.
  • the modified cyclotide is a modified cyclotide that binds to LRP-1 but has altered binding to LRP-1 compared to the cyclotide set forth in any one of SEQ ID NOS: 85-94.
  • the binding affinity to LRP-1 is reduced, such as reduced by 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 10.0-fold or more.
  • the binding affinity to the leptin receptor is pH-dependent, such that the modified cyclotide exhibits greater binding affinity to LRP-1 at neutral pH (e.g. 7.0-7.4) than acidic pH of the endosome (e.g.
  • the modified cyclotide has a loop with a peptide that has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in any one of SEQ ID NOS: 39-48.
  • the modified cyclotide is modified by loop replacement (e.g. loop 1) of Mcoti-II (SEQ ID NO: 2) with a peptide that has 1, 2, 3 or 4 amino acid substitution(s) compared to the peptide set forth in any one of SEQ ID NOS: 39-48.
  • the amino acid substitution(s) is to a histidine.
  • the amino acid substitution(s) is to an alanine.
  • such modified cyclotides exhibit enhanced blood-brain barrier translocation characteristics.
  • any of the provided modified cyclotides bind to a BBB-R for which the peptide targets.
  • Methods for determining binding affinity, or relative binding affinity are known in art, solid-phase ELISA immunoassays, ForteBio Octet, Biacore measurements or flow cytometry. See, for example, Larsen et al., American Journal of Transplantation, vol. 5: 443-453 (2005); Linsley et al., Immunity, Vol 1 (9): 793-801 (1994).
  • binding affinity can be measured by flow cytometry, such as based on a Mean Fluorescence Intensity (MFI) in a flow binding assay.
  • MFI Mean Fluorescence Intensity
  • the provided conjugate such as fusion protein, has a binding affinity for a BBB-R as determined by, for example, solid-phase ELISA immunoassays, flow cytometry or surface plasmon resonance (Biacore) assays.
  • the conjugate (e.g. fusion protein) has a binding affinity for a BBB-R of greater than 10 nM and less than 1000 nM. In some embodiments, the conjugate has a binding affinity for a BBB-R of greater than 20 nM and less than 800 nM.
  • the conjugate has a binding affinity for a BBB-R of about 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM or any value between any of the foregoing. In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 50 nM to about 500 nM.
  • the conjugate has a binding affinity for a BBB-R of from about 50 nM to about 250 nM, In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 50 nM to about 100 nM. In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 100 nM to about 500 nM. In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 100 nM to about 250 nM. In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 250 nM to 500 nM.
  • the cyclotide molecules provided herein may comprise a specific functionality which typically includes at least some type of membrane translocation activity.
  • the membrane translocation activity comprises an activity selected from one or more of: an ability to translocate across the gut wall (e.g. intestinal mucosa); an ability to translocate across the blood brain barrier; an ability to translocate across a cell membrane; an ability to translocate into a sub cellular compartment; an ability to translocate into the nucleus of a cell; and an ability to translocate into an organelle, including a mitochondrion.
  • the provided modified cyclotide exhibits improved or enhanced transcytosis across the blood brain barrier compared to the parental or scaffold cyclotide, such as the cyclotide Mcoti-II (SEQ ID NO:2).
  • the improved or enhanced trancytosis is due to binding to a receptor involved in blood-brain barrier transcytosis.
  • the membrane translocation activity may be suitably comprised within a peptide or polypeptide that resides within the functional module a larger protein.
  • the provided cyclotide represent synthetic polypeptide scaffold. Desirable physical properties of potential scaffold molecules include high thermal stability and reversibility of thermal folding and unfolding.
  • a conjugate comprising: the modified cyclotide or the binding molecule of any of the preceding embodiments, and a biologically active agent.
  • the modified cyclotide or binding molecule is any as described in Section I.B.
  • the biologically active agent is a small molecule, a peptide or a protein.
  • the biologically active agent is a diagnostic agent or a therapeutic agent.
  • the biologicaly active agent is any agent that has therapeutic potential for treatment of pathology in the central nervous system (CNS).
  • the biologically active agent can be selected from eurological disorder drugs, neurotrophic factors, growth factors, enzymes, cytotoxic agents, antibodies directed to a brain target, monoclonal antibodies directed to a brain target, or peptides directed to a brain target.
  • a cyclotide of the invention may also be conjugated or otherwise linked to a biologically active agent or therapeutic selected from the group consisting of: a small molecule; an antibody or antibody fragment; a hormone, a cytokine; a nucleic acid; a bioactive peptide; a glycosylated peptide; an imaging agent; and a radioactive compound.
  • a biologically active agent or therapeutic selected from the group consisting of: a small molecule; an antibody or antibody fragment; a hormone, a cytokine; a nucleic acid; a bioactive peptide; a glycosylated peptide; an imaging agent; and a radioactive compound.
  • the cyclotide moiety can be an amino acid extension of the C- or N-terminus of the biological agent, e.g. polypeptide.
  • a short amino acid linker sequence may lie between the biological agent, e.g. polypeptide, and the cyclotide moiety.
  • Suitable linker groups may comprise an amide, an ester, a disulfide, a sulfide, a ketal, a succinate, an oxime, a carbamate, a carbonate, a sialyl ether, or a triazole.
  • a linker can be a peptide linker.
  • provided molecules include those where the biological agent, e.g. polypeptide, is linked, e.g. by chemical conjugation to the cyclotide moiety, optionally via a linker sequence.
  • the modified polypeptide will be linked to the other moiety via sites that do not interfere with the activity of either moiety.
  • the conjugate is a fusion protein comprising the modified cyclotide operably linked to a biologically active agent that is a protein, polypeptide or peptide.
  • the biologically active agent is selected from the group consisting of an antibody or an antibody fragment thereof. In some of any of the preceding embodiments, the biologically active agent is selected from the group consisting of a growth factor or a hormone. In some of any of the preceding embodiments, the biologically active agent is an enzyme.
  • cyclotides are conjugated to or comprised within desirable biological therapeutic agents which are then administered to a test animal in order to determine the BBB transfer of the modified biological therapeutic agent.
  • Therapeutic uses and applications for the cyclotides of the invention therefore include any disease or condition that requires repetitive treatment regimes or the frequent administration of a biologically active agent. This includes, therapeutic applications that may benefit from conjugation of a cyclotide to the N- or C-terminus of the therapeutic agent that so renders the therapeutic agent more readily available to the brain or cerebrospinal fluid of the recipient.
  • Diseases that are suitable for treatment include but are not limited to: the treatment of various neoplastic and non-neoplastic diseases and disorders (e.g. cancers / neoplastic diseases and related conditions); chronic degenerative and neurodegenerative diseases or disorders (e.g. multiple sclerosis, Parkinson’s disease and Alzheimer’s disease), strokes or other brain damaging conditions.
  • cyclotides of the invention are fused to a therapeutic agent and used as an intravenously administered therapeutic treatment of Alzheimer’s disease or intracranial neoplasms.
  • a cyclotide of the invention is conjugated to a therapeutic agent capable of enhancing cognitive ability such as memory.
  • the present invention relates to a conjugate which may comprise a carrier selected from the group consisting of any one of the modified cyclotides of the present invention linked to a bioactive or therapeutic agent selected from the group consisting, for example, of a drug (e.g., a small molecule drug, e.g., an antibiotic), a medicine, a detectable label, a protein (e.g., an enzyme), protein-based compound (e.g., a protein complex comprising one or polypeptide chain) and a polypeptide (peptide).
  • a drug e.g., a small molecule drug, e.g., an antibiotic
  • a medicine e.g., a medicine
  • a detectable label e.g., a protein (e.g., an enzyme), protein-based compound (e.g., a protein complex comprising one or polypeptide chain) and a polypeptide (peptide).
  • the agent may be more particularly, a molecule which is active at the level
  • the detectable label may be a radio imaging agent.
  • a label which may be conjugated with the carrier of the present invention and which is encompassed herein includes, for example and without limitation, an isotope, a fluorescent label (e.g., rhodamine), a reporter molecule (e.g., biotin), etc.
  • Other examples of detectable labels include, for example, a green fluorescent protein, biotin, a his tag protein and beta-galactosidase.
  • the biologically active agent is a therapeutic protein agent.
  • therapeutic protein or protein-based compound which may be conjugated with the carrier of the present invention and which is encompassed herein includes, without limitation, an antibody, an antibody fragment (e.g., an antibody binding fragment such as a Fv fragment, F(ab)2, F(ab)2' and Fab and the like), a single domain antibody (e.g. such as a camelid VHH domain, a shark novel antigen receptor (NAR), or a human VH or VL domain), a peptidic- or protein-based drug (e.g., a positive pharmacological modulator (agonist) or an pharmacological inhibitor (antagonist)) etc.
  • an antibody e.g., an antibody binding fragment such as a Fv fragment, F(ab)2, F(ab)2' and Fab and the like
  • a single domain antibody e.g. such as a camelid VHH domain, a shark novel antigen receptor (NAR), or a human V
  • agent which are encompassed herein include growth factors (e.g. Fibroblast Growth Factor and related proteins, Nerve Growth Factor, Glial Cell-derived Neurotrophic Factor, Brain-Derived Neurotrophic Factor, Neurotrophin-3 and Neurotrophin-4), cellular toxins (e.g., monomethyl auristatin E (MMAE), toxins from bacteria endotoxins and exotoxins; diphtheria toxins, botunilum toxins, tetanus toxins, pertussis toxins, staphylococcus enterotoxins, toxic shock syndrome toxin TSST-1, adenylate cyclase toxin, shiga toxin, cholera enterotoxin, and others), soluble receptors (such as TNF receptor 1 or 2) and anti -angiogenic compounds (endostatin, catechins, nutriceuticals, chemokine IP- 10, inhibitors of matrix metalloproteinase (MMPIs), anastellin, vironect
  • the biologically active agent is a neurological disorder therapeutic agent.
  • a neurological disorder therapeutic agent includes, but are not limited to, small molecule compounds, antibodies, peptides, proteins, natural ligands of one or more CNS target(s), modified versions of natural ligands of one or more CNS target(s), aptamers, inhibitory nucleic acids (i.e., small inhibitory RNAs (siRNA) and short hairpin RNAs (shRNA)), ribozymes, and small molecules, or active fragments of any of the foregoing.
  • Exemplary neurological disorder drugs of the invention include, but are not limited to: antibodies, aptamers, proteins, peptides, inhibitory nucleic acids and small molecules and active fragments of any of the foregoing that either are themselves or specifically recognize and/or act upon (i.e., inhibit, activate, or detect) a CNS antigen or target molecule.
  • a CNS antigen target molecules includes, but are not limited to, amyloid precursor protein or portions thereof, amyloid beta, beta- secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, glioma or other CNS cancer markers, and neurotrophins.
  • Non-limiting examples of neurological disorder drugs and the corresponding disorders they may be used to treat include brain-derived neurotrophic factor (BDNF) for treating chronic brain injury (Neurogenesis); anti-EGFR antibody for treating Brain cancer; Glial cell-line derived neural factor (GDNF) for treating Parkinson's disease; Brain-derived neurotrophic factor (BDNF) for treating Amyotrophic lateral sclerosis or depression; Lysosomal enzyme for treating Lysosomal storage disorders of the brain; Ciliary neurotrophic factor (CNTF) for treating Amyotrophic lateral sclerosis; Neuregulin-1 for treating Schizophrenia; Anti-HER2 antibody (e.g. trastuzumab) for treating brain metastasis from HER2 -positive cancer.
  • BDNF brain-derived neurotrophic factor
  • GDNF Glial cell-line derived neural factor
  • BDNF Brain-derived neurotrophic factor
  • CNTF Ciliary neurotrophic factor
  • the biologically active agent is an antibody.
  • the antibody is a therapeutic antibody.
  • the antibody is a full length antibody directed to a brain target.
  • the antibody is a full length IgG. Previous studies have illustrated that a very small percentage (approximately 0.1%) of an IgG injected in the bloodstream are able to penetrate into the CNS compartment (Felgenhauer, Klin. Wschr. 52: 1158-1164 (1974)). The provided embodiments thus provide for improved therapeutics with enhanced blood-brain barrier translocation activity.
  • the antibody is directed against, such as specifically binds, an antigen and/or molecule expressed in the CNS, including the brain, which can be targeted with an antibody or small molecule.
  • antigen and/or molecule include, without limitation: beta- seer etase 1 (BACE1), amyloid beta (Abeta), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), Tau, apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), and caspase 6.
  • BACE1 beta- seer etase 1
  • Abeta amyloid beta
  • EGFR epidermal
  • the antibody is directed against or binds HER2.
  • the antibody is trastuzumab (Herceptin®).
  • the antibody contains a heavy chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the heavy chain sequence set forth in SEQ ID NO: 134 and a light chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the light chain sequence set forth in SEQ ID NO: 137.
  • the antibody contains the heavy chain sequence set forth in SEQ ID NO: 134 and the light chain sequence set forth in SEQ ID NO: 137.
  • antibodies targeting HER2, and provided conjugate fusions containing same can be used for treating metastatic breast cancer. In some embodiments, antibodies targeting HER2, and provided conjugate fusions containing same, can be used for treating a neurological disease, such as Alzheimer’s Disease.
  • the antibody is directed against or binds amyloid beta.
  • the antibody is aducanumab (Aduhelm).
  • the antibody contains a heavy chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the heavy chain sequence set forth in SEQ ID NO: 129 and a light chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the light chain sequence set forth in SEQ ID NO: 132.
  • the antibody contains the heavy chain sequence set forth in SEQ ID NO: 129 and the light chain sequence set forth in SEQ ID NO: 132.
  • antibodies targeting amyloid beta, and provided conjugate fusions containing same can be used for treating Alzheimer’s Disease.
  • the biologically active agent is a TNF inhibitor.
  • the inhibitor is a small molecule, peptide or protein.
  • the inhibitor is an antibody.
  • the antibody is directed against or binds TNF-alpha.
  • the antibody is adalimumab (Humira).
  • the antibody contains a heavy chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the heavy chain sequence set forth in SEQ ID NO: 102 and a light chain sequence that exhibits at least about 85%, at least about 90%, or at least about 95% sequence identity to the light chain sequence set forth in SEQ ID NO: 103.
  • the antibody contains the heavy chain sequence set forth in SEQ ID NO: 102 and the light chain sequence set forth in SEQ ID NO: 103.
  • antibodies targeting TNF-alpha, and provided conjugate fusions containing same can be used for treating a neurological disease, such stroke, traumatic brain injury or Alzheimer’s Disease.
  • the biologically active agent is a growth factor or a hormone. In some embodiments, the biologically active agent is a growth factor. Cyclotides of the invention may be operably linked to growth factors including ADNP, BDNF, CNTF, GCSF, GDNF, IFN Beta, IL-10, Insulin, MANF, NGF, NT-3, Progranulin, amongst others in order to treat a variety of neurological disorders such stroke, Alzheimers, Parkinsons disease and multiple sclerosis. In some embodiments, the biologically active agent is a nerve growth factor (NGF), such as for treating Parkinson’s Disease, stroke or Alzheimer’s Disease.
  • NGF nerve growth factor
  • the biologically active agent is granulocyte colony-stimulating factor (GCSF), such as for using in treating stroke or traumatic brain injury (TBI).
  • GCSF granulocyte colony-stimulating factor
  • TBI traumatic brain injury
  • the biologically active agent is IL-10 such as for use in treating Alzheimer’s Disease, heart disease or brain cancer.
  • the biologically active agent is BDNF, such as for use in treating ALS or depression.
  • the biologically active agent is ADNP, such as for use in treating Autism like disorder.
  • the biologically active agent is a NGF and has the sequence set forth in SEQ ID NO: 147 or a sequence that exhibits at least about 85%, at least about 90% or at least about 95% sequence identity to SEQ ID NO: 147. In some embodiments, the biologically active agent is set forth in SEQ ID NO: 147.
  • the biologically active agent is an enzyme.
  • the enzyme is a ceramide degrading enzyme, a lipase, a hydrolase type enzyme or a sulfatase.
  • the enzyme is a ceramide degrading enzyme and the ceramide degrading enzyme is glucocerebrosidase, galactocerebrosidase or alpha galactosidase.
  • the enzyme is a lipase or a hydrolase type enzyme and the enzyme is sphinomyelinase, cerliponase or alpha glucosidase.
  • the enzyme is heparin N Sulfatase, such as for treating Sanfilippo A.
  • the enzyme is glucocerebrosidase, such as for treating Gaucher’s Disease.
  • the enzyme is glucocerebrosidase, such as for treating Parkinson’s Disease.
  • the enzyme is galactocerebrosidase, such as for use in treating Krabbe’s disease.
  • the enzyme is alpha galactosidase, such as for use in treating Fabry’s disease.
  • the enzyme is sphinomyelinase, such as for treating Niemann Pick.
  • the enzyme is cerliponase alpha, such as for use in treating Jansky Bielschowsky.
  • the enzyme is alpha glucosidase, such as for treating Pompe’s.
  • the enzyme is tripeptidyl peptidase I, such as for using in treating Jansky Bieschowsky or Batten.
  • the enzyme is galactosamine 6 sulfatase, such as for use in treating Marquio.
  • the biologically active agent is glucocerebrosidase (GCase).
  • the enzyme has a sequence of amino acids set forth in SEQ ID NO: 144 or a sequence of amino acids that exhibits at least about 85%, at least about 90% or at least about 95% sequence identity to SEQ ID NO: 144. In some embodiments, the sequence is set forth in SEQ ID NO: 144. In some embodiments, the enzyme has a sequence of amino acids set forth in SEQ ID NO: 145 or a sequence of amino acids that exhibits at least about 85%, at least about 90% or at least about 95% sequence identity to SEQ ID NO: 145. In some embodiments, the sequence is set forth in SEQ ID NO: 145.
  • the glucocerebrosidase is a glucocerebrosidase mutant GCase moleucule that exhibits increased stability and enhanced function.
  • An exemplary GCase mutant has one or more amino acid substitions selected from I5N, F31Y, L34Q, M53T, P55T, H145F, H145L, H223N, H223Y, E233Q, H274N, W312C, F316A, L317F, K321V, K321A, K321N, A341C, H365K, I368C, D443C, D445C, H451K, S455C, S464C, R495C, R495N, and combinations thereof, relative to the sequence set forth in SEQ ID NO: 144 or SEQ ID NO: 145.
  • the GCase mutant contains 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions from I5N, F31Y, L34Q, M53T, P55T, H145F, H145L, H223N, H223Y, E233Q, H274N, W312C, F316A, L317F, K321V, K321A, K321N, A341C, H365K, I368C, D443C, D445C, H451K, S455C, S464C, R495C, R495N, relative to the sequence set forth in SEQ ID NO: 144 or SEQ ID NO: 145.
  • the GCase mutant has the amino acid substitutions M53T and P55T, relative to the sequence set forth in SEQ ID NO: 144 or SEQ ID NO: 145. In some embodiments, the GCase mutant has the amino acid substitutions H145L/K321N, D443C/S464C, S455C/R495C, W312C/A341C, I368C/D445C, and E233Q/W312C/A341C, relative to the sequence set forth in SEQ ID NO: 144 or SEQ ID NO: 145.
  • the GCase mutant has the amino acid substitutions F316A/L317F, F316A/L317F/K321N, or H145L/K321N, relative to the sequence set forth in SEQ ID NO: 144 or SEQ ID NO: 145.
  • Non-limiting examples of GCase mutants are described in published PCT Appl. No. WO2012/064709; WO2021/048034; and WO2022/023761.
  • the modified cyclotide is set forth in SEQ ID NO: 101, such as for targeting TrfR.
  • the modified cyclotide is set forth in SEQ ID NO:81, such as for targeting IgFR.
  • the modified cyclotide is set forth in SEQ ID NO:82, such as for targeting IgFR.
  • the modified cyclotide is set forth in SEQ ID NO:85, such as for targeting LRP-1.
  • the modified cyclotide is set forth in SEQ ID NO:87, such as for targeting LRP-1.
  • the modified cyclotide is set forth in SEQ ID NO:89, such as for targeting LRP-1. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:83, such as for targeting RAGE. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 84, such as for targeting RAGE. In some embodiments, the modified cyclotide targets TrfR and the conjugate has the sequence of amino acids set forth in SEQ ID NO: 146 or a sequence of amino acids that has at least 90%, 91%, 92%, 93%, 94%, 05%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 146.
  • the conjugate has the sequence set forth in SEQ ID NO: 146.
  • the modified cyclotide binds to a BBB-R (e.g, TrfR) to transport the glucocerebrosidase across the blood brain barrier.
  • the conjugate such as a fusion protein of a modified cyclotide and a biological protein agent, contains 1, 2, 3, 4, 5 or more copies of the modified cyclotide.
  • the conjugate such as a fusion protein as provided, contains one modified cyclotide such that the conjugate (e.g. fusion protein) exhibits monovalent for binding the BBB-R.
  • the conjugate e.g. fusion protein
  • the conjugate is monovalent for binding any one BBB-R.
  • the conjugate may contain a plurality of copies of the modified cyclotide, such as 2, 3, 4 or 5 copies of the modified cyclotide.
  • the conjugates e.g. fusion proteins
  • a multivalent binding protein comprising a plurality of any of the provided modified cyclotides.
  • each of the modified cyclotides may be linked, directly or indirectly, in tandem on the same polypeptide chain.
  • the fusion protein is a multichain polypeptide and each chain of the polypeptide contains at least one copy of a modified cyclotide.
  • a fusion protein of an antibody e.g. therapeutic antibody
  • a modified cyclotide in which a first heavy chain or light chain of the antibody is linked to a first copy of the modified cyclotide and the second heavy chain or light chain is linked to a second copy of the modified cyclotide.
  • the binding protein is bivalent and contains two copies of the modified cyclotide.
  • the conjugate may contain a plurality of different modified cyclotidse, such as 2, 3, 4 or 5 different modified cyclotides.
  • the conjugates e.g. fusion proteins
  • a multispecific binding protein comprising a plurality of different cyclotides from any of the provided modified cyclotides.
  • the binding protein is multispecific and contains at least two different modified cyclotides that each bind to a different BBB-R.
  • the multispecific binding protein contains 2, 3 or 4 different modified cyclotides in which each modified cyclotides binds a different BBB-R.
  • each of the modified cyclotides may be linked, directly or indirectly, in tandem on the same polypeptide chain.
  • the fusion protein is a multichain polypeptide and each chain of the polypeptide contains a modified cyclotide, which can be different.
  • a fusion protein of an antibody e.g.
  • the binding protein is bispecific and contains two different modified cyclotides that each bind to a different BBB- R.
  • a first modified cyclotide binds to the transferrin receptor and the second modified cyclotide binds to the IGF1R.
  • an intervening span of one or more amino acid residues indirectly covalently bonds the modified cyclotides (e.g. first modified cyclotide and second modified cyclotide) to each other.
  • the linkage can be via the N- terminal to C-terminal residues.
  • the linkage can be made via side chains of amino acid residues that are not located at the N-terminus or C-terminus modified cyclotide.
  • linkages can be made via terminal or internal amino acid residues or combinations thereof.
  • any of a plurality of modified cyclotides can be to a biological agent (or a chain thereof) separated with a peptide linker, such as a flexible linker.
  • the peptide linker may be GGGS or other similar flexible linker, including longer linkers of (GGGS)n where n is 1-3.
  • the conjugate (e.g. fusion protein) containing a plurality of modified cyclotides may include 2, 3, 4 or more modified cyclotides.
  • the modified cyclotides may be the same or different.
  • the modified cycltoides may be the same to produce a bivalent, trivalent or other multivalent molecule.
  • the modified cyclotides may be different to provide a bispecific, trispecific or other multispecific molecule.
  • any of a plurality of modified cyclotides can be linked in tandem with a biological agent in a single polypeptide chain separated with a peptide linker, such as a flexible linker.
  • the peptide linker may be GGGS or other similar flexible linker, including longer linkers of (GGGS)n where n is 1-3.
  • the tandem single polypeptide may include 2, 3, 4 or more modified cyclotides to produce a bivalent, trivalent, tetravalent or other multivalent molecule.
  • the one or more modified cyclotides may be the same or different.
  • the tandem singly polypeptide may include 2, 3, 4 or more different cyclotides to provide a bispecific, trispecific or other multispecific molecule.
  • the fusion protein is composed of an antibody (e.g. therapeutic antibody) and at least one modified cyclotide.
  • the antibody can be linked to 1, 2, 3 or 4 modified cyclotides, any of which may be the same or different.
  • the modified cyclotides can be linked to the heavy chain or the light chain of the antibody.
  • the modified cyclotides can be linked to the N-terminus of a heavy chain or a light chain the antibody.
  • the modified cyclotides can be linked to the C-terminus of a heavy chain or a light chain of the antibody.
  • each of the modified cyclotides are linked to the heavy chain of the antibody.
  • each chain of the antibody is linked to a modified cyclotide.
  • a bivalent fusion protein composed of an antibody (e.g. a therapeutic antibody) and two copies of a modified cyclotide.
  • the modified cyclotide is linked to both heavy chains of the antibody.
  • the modified cyclotide is linked to both light chains of the antibody.
  • the modified cyclotides can be linked to the N-terminus of a heavy chain or a light chain the antibody.
  • the modified cyclotides can be linked to the C-terminus of a heavy chain or a light chain of the antibody. Methods of making antibody fusions are known.
  • the heavy chain and light chain are co-expressed in a cell.
  • a two chain polypeptide when produced in a cell, a two chain polypeptide is formed by dimerization resulting from disulfide formation between two heavy chain molecules.
  • the antibody fusion protein is a homodimer containing two identical copies of the modified cyclotide.
  • a monovalent fusion protein composed of antibody in which there is linked a single modified cyclotide.
  • the antibody fusion protein is monovalent for binding the BBB-R.
  • the modified cyclotide is linked to one heavy chain of the antibody.
  • the modified cyclotide is linked to one light chain of the antibody.
  • the modified cyclotides can be linked to the N-terminus of a heavy chain or a light chain the antibody.
  • the modified cyclotides can be linked to the C-terminus of a heavy chain or a light chain of the antibody.
  • the fusion protein is a heterodimer composed of two different heavy chains and a light chain that pairs with each heavy chain, in which only one of the heavy chains is linked to the modified cyclotide.
  • a multispecific fusion protein composed of an antibody in which there is linked at least two different modified cyclotides.
  • the antibody fusion protein is bispecific and contains two different modified cyclotides for binding two different BBB-R.
  • each modified cyclotide is linked to a heavy chain of the antibody.
  • the modified cyclotides may be linked the a heavy chain in tandem.
  • the modified cyclotides may each be linked to a different heavy chain of the antibody.
  • each modified cyclotide is linked to a light chain of the antibody.
  • the modified cyclotides may be linked the a light chain in tandem.
  • the modified cyclotides may each be linked to a different light chain of the antibody.
  • the modified cyclotides can be linked to the N-terminus of a heavy chain or a light chain the antibody.
  • the modified cyclotides can be linked to the C-terminus of a heavy chain or a light chain of the antibody.
  • the fusion protein is a heterodimer composed of two different heavy chains and a light chain that pairs with each heavy chain, in which one of the heavy chains is linked to different modified cyclotide in tandem and the other heavy chain is not linked to a modified cyclotide.
  • the fusion protein is a heterodimer composed of two different heavy chains and a light chain that pairs with each heavy chain, in which each of the heavy chains is linked to a different modified cyclotide.
  • two different heavy chains may be co-expressed in a cell using knobs- into-hole engineering strategy or other strategy to produce a heterodimer in which two different heavy chains, for example each carrying a different modified cyclotide, may interact to form a heterodimer.
  • residues of the constant chain are modified by amino acid substitution to promote the heterodimer formation.
  • the one more amino acid modifications are selected from a knob-into-hole modification and a charge mutation to reduce or prevent self-association due to charge repulsion.
  • the heterodimer can be formed by transforming into a cell both a first nucleic acid molecule encoding a first heavy chain polypeptide subunit (e.g. knob sequence) and a second nucleic acid molecule encoding a second different heavy chain polypeptide subunit (e.g. hole sequence).
  • the heterodimer is produced upon expression and secretion from a cell as a result of covalent or non-covalent interaction between residues of the two polypeptide subunits to mediate formation of the dimer.
  • a mixture of dimeric molecules is formed, including homodimers and heterodimers. For the generation of heterodimers, additional steps for purification can be necessary.
  • the first and second polypeptide can be engineered to include a tag with metal chelates or other epitope, where the tags are different.
  • the tagged domains can be used for rapid purification by metal-chelate chromatography, and/or by antibodies, to allow for detection by western blots, immunoprecipitation, or activity depletion/blocking in bioassays. In other embodiments, methods can be carried out that promote heterodimerization.
  • Methods include those described in U.S. Patent No. 10,995,127.
  • having an amino acid modification within the CH3 domain at Thr366, which when replaced with a more bulky amino acid, e.g., Try (T366W) is able to preferentially pair with a second CH3 domain having amino acid modifications to less bulky amino acids at positions Thr366, Leu368, and Tyr407, e.g., Ser, Ala and Vai, respectively (T366S/L368A/Y407V).
  • the “knob” Fc domain comprises the mutation T366W.
  • the “hole” Fc domain comprises mutations T366S, L368A, and Y407V.
  • Fc domains used for heterodimerization comprise additional mutations, such as the mutation S354C on a first member of a heterodimeric Fc pair that forms an asymmetric disulfide with a corresponding mutation Y349C on the second member of a heterodimeric Fc pair.
  • one member of a heterodimeric Fc pair comprises the modification H435R or H435K to prevent protein A binding while maintaining FcRn binding.
  • one member of a heterodimeric Fc pair comprises the modification H435R or H435K, while the second member of the heterodimeric Fc pair is not modified at H435.
  • the hole Fc domain comprises the modification H435R or H435K (referred to as “hole-R” in some instances when the modification is H435R), while the knob Fc domain does not.
  • the hole-R mutation improves purification of the heterodimer over homodimeric hole Fc domains that may be present.
  • knob and hole sequence of the exemplary antibody trastuzumab is set forth in SEQ ID NO: 135 and SEQ ID NO: 136, respectively.
  • a knob and hole sequence of the exemplary antibody aducanumab is set forth in SEQ ID NO: 130 and SEQ ID NO: 131, respectively.
  • Any antibody can be similarly modified in the CH3 domain to create knob and hole chains to promote heterodimerization.
  • a modified cyclotide is linked to the knob heavy chain and/or a hole heavy chain of an antibody.
  • the knob heavy chain, hole heavy chain and light chain are co-expressed in a cell in which a heterodimer antibody containing two different heavy chains (knob and hole) each complexed with the light chain is produced.
  • one or more “peptide linkers” link the modified cyclotide and one or more other modified cyclotide or the biological agent.
  • a peptide linker can be a single amino acid residue or greater in length.
  • the peptide linker has at least one amino acid residue but is no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in length.
  • the pin some embodiments, the linker is a flexible linker.
  • the linker is (in one-letter amino acid code): GGGGS (“4GS”) or multimers of the 4GS linker, such as repeats of 2, 3, 4, or 5 4GS linkers.
  • the linker is or includes GGGGS (SEQ ID NO: 148).
  • the peptide linker is or includes (GGGGS)2 or (GGGGS)s as set forth in SEQ ID NOs: 154 and 155, respectively.
  • the linker also can include a series of alanine residues alone or in addition to another peptide linker (such as a 4GS linker or multimer thereof).
  • the linker is GSGGGS GGGGS GGGGS (SEQ ID NO: 104).
  • the linker is a GS linker of at least 10 amino acids in length, such as at least 15 amino acids in length.
  • the GS linker is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length, or any value between any of the foregoing.
  • the GS linker is 10 to 25 amino acids.
  • the GS linker is 10 to 20 amino acids.
  • the GS linker is GGSGGSGGSGGS, i.e., (GGS)4 (SEQ ID NO: 186); GGSGGSGGSGGSGGS, i.e., (GGS)5 (SEQ ID NO: 187); GGGGGSGGGGGSGGGGGS, i.e., (G5S)3 (SEQ ID NO: 188), GGSGGGGSGGGGSGGGGS (SEQ ID NO: 189) and GGGGS GGGGS GGGGS (SEQ ID NO: 155), or GSGGGSGGGGSGGGGS (SEQ ID NO: 104).
  • the GS linker is set forth in SEQ ID NO: 104.
  • the linker is a cleavable linker.
  • the use of a cleavable linker can be used to ensure release of the free protein, i.e. biological agent (e.g. therapeutic agent) to the brain.
  • cleavable linkers include the acid labile linkers. Acid labile linkers include cis-aconitic acid, cis-carboxylic alkadienes , ciscarboxylic alkatrienes , and poly-maleic anhydrides. Other cleavable linkers are linkers capable of attaching to primary alcohol groups.
  • the linker is a cleavable linker that contains an endosomespecific protease cleavage site.
  • the endosome-specific protease cleavage site is a cathepsin cleavage site and the linker is a cathepsin cleavable linker.
  • the cathespin is a cathepsin B, a cathepsin D, a cathepsin K, a cathepsin S or a cathepsin L.
  • the cathepsin cleavage site may be any as described in PCT publication No. WO2016/050934.
  • the linker containing a cathepsin cleavage site is set forth in any one of SEQ ID NOS: 133 and 156-176. In some embodiments, the linker has a cathepsin B cleavage site. In some embodiments, the cathepsin B linker is set forth in SEQ ID NO:133.
  • the provided conjugates is a bivalent cyclotide antibody fusion composed of an antibody linked to a modified cyclotide, such as any modified cyclotide described herein.
  • the modified cyclotide is linked to the C-terminus of both heavy chains of the antibody.
  • the antibody is adalimumab.
  • the antibody fusion contains a heavy chain composed of the adalimumab heavy chain set forth in SEQ ID NO: 102, a linker and a modified cyclotide, such as any described in Section I.B; and a light chain set forth in SEQ ID NO: 103.
  • the antibody fusion contains a heavy chain composed of the trastuzumab heavy chain set forth in SEQ ID NO: 103, a linker and a modified cyclotide, such as any described in Section I.B; and a light chain set forth in SEQ ID NO: 137.
  • the antibody is a homodimer composed of two identical heavy chains and two identical light chains.
  • the modified cyclotide is set forth in SEQ ID NO:72, such as for targeting TrfR.
  • the modified cyclotide is set forth in SEQ ID NO: 101, such as for targeting TrfR.
  • the modified cyclotide is set forth in SEQ ID NO:81, such as for targeting IgFR. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:82, such as for targeting IgFR. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:85, such as for targeting LRP-1. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:87, such as for targeting LRP-1. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:89, such as for targeting LRP-1. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:83, such as for targeting RAGE.
  • the modified cyclotide is set forth in SEQ ID NO:84, such as for targeting RAGE.
  • the linker is a GS linker of at least 10 amino acids in length, such as at least 15 amino acids in length. In some embodiments the GS linker is 10 to 25 amino acids. In some embodiments, the GS linker is set forth in SEQ ID NO: 104. Also provided herein are antibody fusion conjugates having a sequence that has at least 90%, 91%, 92%, 93%, 94%, 05%, 96%, 97%, 98%, 99% or more sequence identity to any of the foregoing amino acid sequences. In some embodiment, the modified cyclotide binds to the BBB-R recognized by the modified cyclotide to to transport the antibody across the blood brain barrier.
  • the provided conjugates is a monovalent cyclotide antibody fusion composed of an antibody linked to a modified cyclotide, such as any modified cyclotide described herein.
  • the modified cyclotide is linked to the C-terminus of one heavy chain of a heterodimeric antibody.
  • the antibody fusion contains a knob heavy chain of the antibody; a second heavy chain composed of a hole heavy chain of the antibody; and a light chain of the antibody, in which either the knob heavy chain or the hole heavy chain is linked at its C-terminus by a linker to a modified cyclotide, such as any described in Section I.B.
  • the antibody can be a heterodimeric antibody (e.g., knob and hole variant) of any desired therapeutic or diagnostic antibody desired for transport across the blood-brain barrier.
  • Non-limiting examples of antibodies include any as described herein.
  • the antibody is aducanumab.
  • the antibody fusion contains a first heavy chain that is a hole aducanumab heavy chain set forth in SEQ ID NO: 131; a second heavy chain composed of a knob aducanumab heavy chain set forth in SEQ ID NO: 130, a linker and a modified cyclotide, such as any described in Section I.B; and a light chain set forth in SEQ ID NO: 132.
  • the antibody fusion contains a first heavy chain composed of a aducanumab hole heavy chain set forth in SEQ ID NO: 131, a linker, and a modified cyclotide, such as any described in Section I.B; a second heavy chain that is a knob aducanumab heavy chain set forth in SEQ ID NO: 130; and a light chain set forth in SEQ ID NO: 132.
  • the antibody fusion contains a first heavy chain that is a trastuzumab hole heavy chain set forth in SEQ ID NO: 136 or 178; a second heavy chain composed of a knob tratuzumab heavy chain set forth in SEQ ID NO: 135, a linker and a modified cyclotide, such as any described in Section I.B; and a light chain set forth in SEQ ID NO: 137.
  • the modified cyclotide is set forth in SEQ ID NO:72, such as for targeting TrfR.
  • the modified cyclotide is set forth in SEQ ID NO: 101, such as for targeting TrfR.
  • the modified cyclotide is set forth in SEQ ID NO:81, such as for targeting IgFR. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:82, such as for targeting IgFR. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:85, such as for targeting LRP-1. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:87, such as for targeting LRP-1. In some embodiments, the modified cyclotide is set forth in SEQ ID NO:89, such as for targeting LRP-1. In some embodiments, the modified cyclotide is set forth in SEQ ID NO: 83, such as for targeting RAGE.
  • the modified cyclotide is set forth in SEQ ID NO:84, such as for targeting RAGE.
  • the linker is a GS linker of at least 10 amino acids in length, such as at least 15 amino acids in length. In some embodiments the GS linker is 10 to 25 amino acids. In some embodiments, the GS linker is set forth in SEQ ID NO: 104. Also provided herein are antibody fusion conjugates having a sequence that has at least 90%, 91%, 92%, 93%, 94%, 05%, 96%, 97%, 98%, 99% or more sequence identity to any of the foregoing amino acid sequences. In some embodiment, the modified cyclotide binds to the BBB-R recognized by the modified cyclotide to to transport the antibody across the blood brain barrier.
  • conjugates is a bispecific cyclotide antibody fusion composed of an antibody linked to two different modified cyclotides, such as any modified cyclotide described herein.
  • one modified cyclotide is linked to the C- terminus of one heavy chain of a heterodimeric antibody and the other modified cyclotide is linked ot the C-terminus of the other heavy chain of the heterodimeric antibody.
  • the antibody fusion contains a knob heavy chain of the antibody linked at its C-terminus by a linker to a first modified cyclotide; a second heavy chain composed of a hole heavy chain of the antibody linked at its C-terminus by a linker to a second modified cyclotide; and a light chain of the antibody.
  • the antibody can be a heterodimeric antibody (e.g., knob and hole variant) of any desired therapeutic or diagnostic antibody desired for transport across the blood-brain barrier.
  • Non-limiting examples of antibodies include any as described herein.
  • the antibody is aducanumab.
  • the antibody fusion contains a first heavy chain that is a hole aducanumab heavy chain set forth in SEQ ID NO: 131, a linker and a first modified cyclotide; a second heavy chain composed of a knob aducanumab heavy chain set forth in SEQ ID NO: 130, a linker and a second modified cyclotide; and a light chain set forth in SEQ ID NO: 132.
  • the antibody fusion contains a first heavy chain that is a hole trastuzumab heavy chain set forth in SEQ ID NO: 136, a linker and a first modified cyclotide; a second heavy chain composed of a knob trastuzumab heavy chain set forth in SEQ ID NO: 135, a linker and a second modified cyclotide; and a light chain set forth in SEQ ID NO: 137.
  • the first and second modified cyclotide are different and are each independently any as described in Section I.B.
  • modified cyclotides are independently selected from a modified cyclotide that targets TrfR (e.g., set forth in SEQ ID NO:72 or SEQ ID NO: 101), targets IgFR (e.g, set forth in SEQ ID NO:81 or SEQ ID NO:82), targets LRP-1 (e.g., SEQ ID NO:85, SEQ ID NO:87 or SEQ ID NO:89), or for targeting RAGE (e g., SEQ ID NO:83 or SEQ ID NO:84).
  • TrfR e.g., set forth in SEQ ID NO:72 or SEQ ID NO: 101
  • targets IgFR e.g, set forth in SEQ ID NO:81 or SEQ ID NO:82
  • targets LRP-1 e.g., SEQ ID NO:85, SEQ ID NO:87 or SEQ ID NO:89
  • RAGE e., SEQ ID NO:83 or SEQ ID NO:84.
  • one of the first and second modified cyclotide is the modified cyclotide set forth in SEQ ID NO:72 and the other of the first and second modified cyclotide is the cyclotide set forth in SEQ ID NO:82.
  • one of the first and second modified cyclotide is the modified cyclotide set forth in SEQ ID NO: 101 and the other of the first and second modified cyclotide is the cyclotide set forth in SEQ ID NO:82.
  • the linker is a GS linker of at least 10 amino acids in length, such as at least 15 amino acids in length.
  • the GS linker is 10 to 25 amino acids. In some embodiments, the GS linker is set forth in SEQ ID NO: 104. Also provided herein are antibody fusion conjugates having a sequence that has at least 90%, 91%, 92%, 93%, 94%, 05%, 96%, 97%, 98%, 99% or more sequence identity to any of the foregoing amino acid sequences. In some embodiment, the modified cyclotide binds to the BBB-R recognized by the modified cyclotide to to transport the antibody across the blood brain barrier.
  • any of the provided conjugates such as fusion proteins, bind to a BBB-R, which binding is conferred by the peptide of the modified cyclotide of the conjugate.
  • Methods for determining binding affinity, or relative binding affinity are known in art, solid-phase ELISA immunoassays, ForteBio Octet, Biacore measurements or flow cytometry. See, for example, Larsen et al., American Journal of Transplantation, vol. 5: 443- 453 (2005); Linsley et al., Immunity, Vol 1 (9): 793-801 (1994).
  • binding affinity can be measured by flow cytometry, such as based on a Mean Fluorescence Intensity (MFI) in a flow binding assay.
  • MFI Mean Fluorescence Intensity
  • the provided conjugate, such as fusion protein has a binding affinity for a BBB-R as determined by, for example, solidphase ELISA immunoassays, flow cytometry or surface plasmon resonance (Biacore) assays.
  • the conjugate (e.g. fusion protein) has a binding affinity for a BBB-R of greater than 10 nM and less than 1000 nM. In some embodiments, the conjugate has a binding affinity for a BBB-R of greater than 20 nM and less than 800 nM.
  • the conjugate has a binding affinity for a BBB-R of about 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM or any value between any of the foregoing. In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 50 nM to about 500 nM.
  • the conjugate has a binding affinity for a BBB-R of from about 50 nM to about 250 nM, In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 50 nM to about 100 nM. In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 100 nM to about 500 nM. In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 100 nM to about 250 nM. In some embodiments, the conjugate has a binding affinity for a BBB-R of from about 250 nM to 500 nM. In some embodiments, the binding affinity is about 50 nM to 500 nM.
  • the conjugate e.g. fusion protein
  • a cleavable linker such as an endosome-specific cleavable linker (e.g. cathspein cleavable linker)
  • the conjugate can have a higher binding affinity for the BBB-R.
  • the conjugate e.g. fusion protein
  • the conjugate containing a modified cyclotide exhibits improved or enhanced delivery of the bioactive or therapeutic agent to the brain compared to an unconjugated form of the bioactive or therapeutic agent that is not conjugated to the modified cyclotide.
  • the delivery is improved or enhanced by about 1.2-fold, about 1.3-fold, 1.4-fold, 1.5-fold, 1.8-fold, 2.0-fold, 2.5-fold, 3.0-fold, 3.5-fold, 4.0- fold, 4.5-fold, 5.0-fold, 5.5-fold or more.
  • a conjugate as provided delivers greater than 0.1% of the bioactive or therapeutic agent injected into the bloodstream (e.g., by subcutaneous or intravenous injection) to the brain.
  • a conjugate as provided delivers greater than 0.2%, greater than 0.25%, greater than 0.3%, greater than 0.35%, greater than 0.40% or more into the brain.
  • a further embodiment provided herein provides for a nucleic acid sequence that encodes the amino acid sequence of any of the provided binding molecules, such as any of the provided modified cyclotides or fusion proteins containing the same.
  • the nucleic acid sequence may be comprised within a vector, suitably an expression vector, optionally a display vector (including phage or cis-display vectors), where the encoding DNA is operably linked to the peptide.
  • the methods include culturing a host cells under conditions permissive for expression of the fusion protein. In some embodiments, the provided methods further include, following the culturing, isolating the expressed modified cyclotide or fusion protein from the supernatant or from a lysate of the host cell.
  • the nucleic acids encoding the binding molecule can be introduced into cells using recombinant DNA and cloning techniques.
  • a recombinant DNA molecule encoding a polypeptide is prepared. Methods of preparing such DNA molecules are well known in the art.
  • the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidite method.
  • a recombinant or synthetic nucleic acid may be generated through polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the DNA can be cloned into an appropriate transduction/transfection vector as is known to those of skill in the art. Also provided are expression vectors containing the nucleic acid molecules.
  • the expression vectors are capable of expressing the binding molecules, such as modified cyclotides or fusion proteins containing the same, in an appropriate cell under conditions suited to expression of the protein.
  • nucleic acid molecule or an expression vector comprises the DNA molecule that encodes the modified cyclotide or fusion protein operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known.
  • Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.
  • vectors that include any of the nucleic acid sequence encoding a binding molecule, such as a modified cyclotide or fusion protein.
  • Vectors suitable for use include those commonly used in genetic engineering technology, such as bacteriophages, plasmids, cosmids, viruses, or retroviruses.
  • Vectors suitable for use may include other expression control elements, such as a transcription starting site, a transcription termination site, a ribosome binding site, a RNA splicing site, a polyadenylation site, a translation termination site, etc.
  • Vectors suitable for use i may further include additional regulatory elements, such as transcription/translation enhancer sequences, and at least a marker gene or reporter gene allowing for the screening of the vectors under suitable conditions.
  • Marker genes suitable for use include, for instance, dihydrofolate reductase gene and G418 or neomycin resistance gene useful in eukaryotic cell cultures, and ampicillin, streptomycin, tetracycline or kanamycin resistance gene useful in E. coli and other bacterial cultures.
  • Vectors suitable for use in this invention may further include a nucleic acid sequence encoding a secretion signal. These sequences are well known to those skilled in the art.
  • the expression vector further includes a promoter sequence to control the expression of the modified cyclotide or fusion protein.
  • promoter sequence refers to a DNA sequence, which is generally located upstream of a gene present in a DNA polymer, and provides a site for initiation of the transcription of said gene into mRNA.
  • Promoter sequences suitable for use may be derived from viruses, bacteriophages, prokaryotic cells or eukaryotic cells, and may be a constitutive promoter or an inducible promoter.
  • the promoter sequence is operably linked to the sequence encoding the modified cyclotide or fusion protein.
  • operatively linked means that a first sequence is disposed sufficiently close to a second sequence such that the first sequence can influence the second sequence or regions under the control of the second sequence.
  • a promoter sequence may be operatively linked to a gene sequence, and is normally located at the 5'-terminus of the gene sequence such that the expression of the gene sequence is under the control of the promoter sequence.
  • a regulatory sequence may be operatively linked to a promoter sequence so as to enhance the ability of the promoter sequence in promoting transcription. In such case, the regulatory sequence is generally located at the 5'-terminus of the promoter sequence.
  • Promoter sequences suitable for use include any one of the following: viruses, bacterial cells, yeast cells, fungal cells, algal cells, plant cells, insect cells, animal cells, and human cells.
  • a promoter useful in bacterial cells includes, but is not limited to, tac promoter, T7 promoter, T7 Al promoter, lac promoter, trp promoter, trc promoter, araBAD promoter, and Z.PR.PL promoter.
  • a promoter useful in plant cells includes, e.g., 35S CaMV promoter, actin promoter, ubiquitin promoter, etc.
  • Regulatory elements suitable for use in mammalian cells include CMV-HSV thymidine kinase promoters, SV40, RSV- promoters, CMV enhancers, or SV40 enhancers.
  • the recombinant gene product (protein) produced may either remain within the recombinant cell, be secreted into the culture medium, be secreted into periplasm, or be retained on the outer surface of a cell membrane.
  • the recombinant gene product (protein) produced by the method can be purified by using a variety of standard protein purification techniques, including, but not limited to, affinity chromatography, ion exchange chromatography, gel filtration, electrophoresis, reverse phase chromatography, chromatofocusing and the like.
  • the recombinant gene product (protein) produced by the method is preferably recovered in "substantially pure” form. As used herein, the term “substantially pure” refers to a purity of a purified protein that allows for the effective use of said purified protein as a commercial product.
  • the provided methods for producing a binding molecule can be performed using any host organism or cell which is capable of expressing heterologous polypeptides, and is capable of being genetically modified.
  • a host organism will be used herein throughout, but it should be understood, that a host organism can be substituted for the host cell, unless unfeasible for technical reasons.
  • the host cells can be a variety of eukaryotic cells, such as in yeast cells, or with mammalian cells such as Chinese hamster ovary (CHO) or HEK293 cells.
  • the host cell is a suspension cell and the polypeptide is engineered or produced in cultured suspension, such as in cultured suspension CHO cells, e.g. CHO-S cells.
  • the cell line is a CHO cell line that is deficient in DHFR (DHFR-), such as DG44 and DUXB11.
  • the cell is deficient in glutamine synthase (GS), e.g.
  • the CHO cells such as suspension CHO cells, may be CHO-S-2H2 cells, CHO-S-clone 14 cells, or ExpiCHO-S cells.
  • host cells can also be prokaryotic cells, such as with E. coli.
  • the transformed recombinant host is cultured under polypeptide expressing conditions, and then purified to obtain a soluble protein.
  • Recombinant host cells can be cultured under conventional fermentation conditions so that the desired polypeptides are expressed. Such fermentation conditions are well known in the art.
  • the polypeptides provided herein can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, and affinity chromatography. Protein refolding steps can be used, as desired, in completing configuration of the mature protein.
  • HPLC high performance liquid chromatography
  • polypeptides provided herein can also be made by synthetic methods.
  • Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides.
  • well known solid phase synthesis techniques include the use of protecting groups, linkers, and solid phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, use of scavengers, and other aspects of solid phase peptide synthesis. Peptides can then be assembled into the polypeptides as provided herein.
  • a pharmaceutical composition comprising any of the providing molecules of any of the preceding embodiments, including any of the modified cyclotides or conjugates or fusion proteins containing the same, and a pharmaceutically acceptable excipient.
  • said pharmaceutical composition is used for the treatment of a neurological disease.
  • said pharmaceutical composition is used for the diagnosis of a neurological disease.
  • compositions typically contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Suitable examples of such carriers or diluents include, but are not limited to, water, saline, ringer’s solutions, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • the medicaments and pharmaceutical compositions of the invention can take the form of liquids, solutions, suspensions, lotions, gels, tablets, pills, pellets, powders, modified- release formulations (such as slow or sustained-release), suppositories, emulsions, aerosols, sprays, capsules (for example, capsules containing liquids or powders), liposomes, microparticles or any other suitable formulations known in the art.
  • suitable pharmaceutical vehicles are described in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, see for example pages 1447- 1676.
  • surfactant might be added as a wetting agent.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
  • Nonionic detergents that could be included in the formulation as surfactants include: lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants, when used, could be present in the formulation of the peptide or nucleic acid or derivative either alone or as a mixture in different ratios.
  • Additives may be included to further enhance cellular uptake of the cyclotide of the invention, such as the fatty acids oleic acid, linoleic acid and linolenic acid.
  • a pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intratumoral, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • the compositions are formulated for parenteral administration.
  • the compositions are formulated for subcutaneous or intravenous administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. These pharmaceutical compositions can be included in diagnostic kits with instructions for use.
  • compositions are administered in an amount effective for treatment or prophylaxis of the specific indication.
  • the therapeutically effective amount is typically dependent on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated.
  • the pharmaceutical composition may be administered in an amount in the range of about 50 pg/kg body weight to about 50 mg/kg body weight per dose.
  • the pharmaceutical composition may be administered in an amount in the range of about 100 pg/kg body weight to about 50 mg/kg body weight per dose.
  • the pharmaceutical composition may be administered in an amount in the range of about 100 pg/kg body weight to about 20 mg/kg body weight per dose.
  • the pharmaceutical composition may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.
  • the pharmaceutical composition may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the pharmaceutical composition may be administered in an amount in the range of about 20 mg to about 200 mg per dose.
  • the pharmaceutical composition may be administered as needed to subjects.
  • an effective dose of the pharmaceutical composition is administered to a subject one or more times.
  • an effective dose of the pharmaceutical composition is administered to the subject once a month, less than once a month, such as, for example, every two months, every three months, or every six months.
  • an effective dose of the pharmaceutical composition is administered more than once a month, such as, for example, every two weeks, every week, twice per week, three times per week, daily, or multiple times per day.
  • An effective dose of the pharmaceutical composition is administered to the subject at least once.
  • the effective dose of the pharmaceutical composition may be administered multiple times, including for periods of at least a month, at least six months, or at least a year.
  • the pharmaceutical composition is administered to a subject as-needed to alleviate one or more symptoms of a condition.
  • Also provided herein in some embodiments is a method for transporting a biologically active agent across a blood brain barrier of an individual, the method comprising administering the conjugate (e.g., fusion protein) or the pharmaceutical composition of any of the preceding embodiments to a mammal in need thereof.
  • the conjugate e.g., fusion protein
  • the pharmaceutical composition of any of the preceding embodiments
  • conjugates e.g., fusion protein
  • pharmaceutical compositions comprising the same are administered to a subject in an effective amount to effect treatment of the neurological disease.
  • the methods are carried out by administering the conjugates (e.g., fusion protein), or compositions comprising the same, to the subject having, having had, or suspected of having the neurological disease. In some embodiments, the methods thereby treat the neurological disease in the subject. Also provided herein are of use of any of the compositions, such as pharmaceutical compositions provided herein, for the treatment of a neurological disease.
  • Also provided herein in some embodiments is a method for diagnosing a neurological disease in a patient in need thereof comprising administering the conjugate or the pharmaceutical composition of any of the preceding embodiments to said patient and wherein said conjugate comprises a radiolabel.
  • the mammal has a neurological disease.
  • the neurological disease is selected from the group consisting of Alzheimer's disease (AD), stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman's syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, cancer, and traumatic brain injury.
  • said neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson's disease, stroke, a brain tumor, a brain metastasis or a traumatic brain injury (TBI).
  • TBI traumatic brain injury
  • said neurological disease is a congenital disease that is selected from the group consisting of Austen Disease, Canavan Disease, Gaucher’s Disease, Hunter Syndrome, Hurler-Scheie Syndrome, Jansky Bielschowsky Disease, Krabbe Disease, LCAT Deficiency, Lowe Syndrome, Maroteaux-Lamy Syndrome, Morquio Syndrome A, Morquio Syndrome B, Sanfilippo Syndrome A, Sanfilippo Syndrome B, Sanfilippo Syndrome C, Sanfilippo Syndrome D, Spinal Muscular Atrophy, Tay Sachs Disease, and Walker-Warburg Syndrome.
  • the neurological disease includes non-neoplastic diseases and disorders, such as cancers / neoplastic diseases and related conditions).
  • the neurological disease is an intracranial neoplasms.
  • the neurological disease or disorder is for treatment of strokes or other brain damaging conditions.
  • a cyclotide of the invention is conjugated to a therapeutic agent capable of enhancing cognitive ability such as memory.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a glucocerebrosidase is administered to a subject for treating a neurodegenerative disease.
  • the neurological disease is Gaucher’s disease.
  • the neurological disease is Parkison’s Disease.
  • the glucocerebrosidase and conjugates containing the same can be any as descried in Section II.
  • Glucocerebrosidase is a lysosomal luminal membrane-associated hydrolase, where it interacts with glucolipids.
  • GBA glucocerebrosidase
  • GD Gaucher disease
  • the neurological disease is Gaucher’s disease.
  • Symptoms of GD include anemia, enlargement of liver and spleen, bone lesions and in severe cases neurologic manifestations, widely classified into three clinical subtypes (Beutler et al, 1994; Komhaber et al, 2008). The most common form is classified into type I Gaucher disease with nearly no neuronopathic implications, currently treatable with the use of enzyme replacement therapy (ERT).
  • Type II and III are more severe types of GD with type II having acute neuronopathic phenotype, indicating symptoms at near birth, progressing until death during early infancy.
  • Type III causes chronic severe neuropathic symptoms, including learning disabilities, cardiac abnormalities and myoIonic epilepsy (Beutler et al, 1994; Sidransky et al, 2007; Sidransky & Lopez, 2012).
  • the provided methods are for treating type I Gaucher disease.
  • the provided methods are for treating type II Gaucher disease.
  • the provided methods are for treating type III Gaucher disease.
  • the subject to be treated has a mutation in the natural glucocerebrosidase gene (also called GCase or GBA gene), which is associated with the neurological disease, such as GD, in the subject.
  • a mutation in the natural glucocerebrosidase gene also called GCase or GBA gene
  • GBA gene the natural glucocerebrosidase gene
  • GD the neurological disease
  • Approximately, 300 mutations have been identified and directly linked to the progression of GD, resulting in a wide pathological spectrum of this disorder (Alfonso et al, 2007; Grabowski & Horowitz, 1997). Inherited missense and nonsense mutations in the GBA gene can lead to misfolding, mistrafficking and destabilization of the glucocerebrosidase enzyme.
  • the subject carries the GCase mutation N370S and/or L444P.
  • Patients carrying the mutated GCase variant N370S are usually diagnosed with type I GD, possessing a wide range of symptoms.
  • N370S is responsible for conserving and stabilizing proper conformation of the active binding pocket (Lieberman et al , 2007).
  • L444P reveals a more severe type categorized in type II or III when carrying this GBA gene mutation, possibly disrupting the hydrophobic structure, altering the domain II function (Lieberman et al, 2007).
  • a conjugate e.g. fusion protein
  • a conjugate containing a modified cyclotide linked to an anti-HER.2 antibody is administered to a subject for treating a neurological disease or disorder.
  • the anti-HER2 antibody and conjugates containing the same can be any as described in Section II.
  • the anti-HER2 antibody is trastuzumab.
  • the conjugate is used for treating a metastatic brain cancer.
  • the conjugates are used for treating Alzheimer’s Disease.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to an anti-TNF-alpha antibody is administered to a subject for treating a neurological disease or disorder.
  • the anti-TNF-alpha antibody and conjugates containing the same can be any as described in Section II.
  • the anti-TNF-alpha antibody is adalimumab.
  • the neurological disease or disorder is a stroke, traumatic brain injury or Alzheimer’s Disease.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to an anti-amyloid beta antibody is administered to a subject for treating a neurological disease or disorder.
  • the anti-amyloid beta antibody and conjugates containing the same can be any as described in Section II.
  • the anti -amyloid beta antibody is aducanumab.
  • the neurological disease or disorder is Alzheimer’s Disease.
  • a conjugate e.g. fusion protein
  • NGF nerve growth factor
  • a conjugate containing a modified cyclotide linked to an nerve growth factor (NGF) is administered to a subject for treating a neurological disease or disorder.
  • the NGF and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Parkinson’s Disease, stroke or Alzheimer’s Disease.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a granulocyte colony-stimulating factor is administered to a subject for treating a neurological disease or disorder.
  • the GCSF and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is stroke or a traumatic brain injury.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a interleukin 10 IL-10
  • IL-10 and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Alzheimer’s Disease.
  • a conjugate e.g. fusion protein
  • BDNF brain-derived neurotrophic factor
  • a subject for treating a neurological disease or disorder.
  • the BDNF and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is ALS or depression.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a activity-dependent neuroprotective protein is administered to a subject for treating a neurological disease or disorder.
  • the ADNP and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is an autism-like disorder.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a heparin N Sulfatase is administered to a subject for treating a neurological disease or disorder.
  • the heparin N Sulfatase and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Sanfilippo A.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a galactocerebrosidase is administered to a subject for treating a neurological disease or disorder.
  • the galactocerebrosidase and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Krabbe’s disease.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to an alpha galactosidase is administered to a subject for treating a neurological disease or disorder.
  • the alpha galactosidase and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Fabry’s disease.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a sphinomyelinase is administered to a subject for treating a neurological disease or disorder.
  • the sphinomyelinase and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Niemann Pick.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a cerliponase alpha is administered to a subject for treating a neurological disease or disorder.
  • the cerliponase alpha and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Jansky Bielschowsky.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a alpha glucosidase is administered to a subject for treating a neurological disease or disorder.
  • the alpha glucosidase and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Pompe’s.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a tripeptidyl peptidase I is administered to a subject for treating a neurological disease or disorder.
  • the tripeptidyl peptidase I and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Jansky Bieschowsky or Batten.
  • a conjugate e.g. fusion protein
  • a modified cyclotide linked to a galactosamine 6 sulfatase is administered to a subject for treating a neurological disease or disorder.
  • the galactosamine 6 sulfatase and conjugates containing the same can be any as described in Section II.
  • the neurological disease or disorder is Marquio syndrome.
  • the methods and uses include administering a provided conjugate (e.g., fusion protein) or a pharmaceutical composition comprising the same into a subject (e.g., a human).
  • a provided conjugate e.g., fusion protein
  • a pharmaceutical composition comprising the same into a subject (e.g., a human).
  • the administeration to the subject by a parenteral administration e.g., the administration is intramuscularly, subcutaneously, intravenously, topically, orally or by inhalation.
  • the administration is intramuscular.
  • the administration is subcutaneous.
  • a provided conjugate e.g., fusion protein
  • the effective or therapeutically effective dose is a dose for treating a neurological disorder.
  • the effective or therapeutically effective dose is an amount sufficient to alleviate one or more signs and/or symptoms of the neurological disorder in the treated subject, whether by inducing the regression or elimination of such signs and/or symptoms or by inhibiting the progression of such signs and/or symptoms.
  • the dose amount may vary depending upon the age and the size of a subject to be administered, target disease, conditions, route of administration, and the like.
  • an effective or therapeutically effective dose of a provided conjugate (e.g., fusion protein) for treating a neurological disorder, e.g., in an adult human subject is about 0.001 mg/kg to about 200 mg/kg, such as 0.01 mg/kg to 200 mg/kg or 0.1 mg/kg to 200 mg/kg.
  • a provided conjugate e.g., fusion protein
  • the frequency and the duration of the treatment can be adjusted.
  • the cyclotide-conjugated therapeutic molecules of the present invention are utilized as separately administered compositions or in conjunction with other therapeutic agents.
  • additional agents can include various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins, or chemotherapeutic drugs such as tamoxifen, paclitaxel, oxaliplatin, vincristine and fluorouracil.
  • Pharmaceutical compositions can include combinations of various cytotoxic or other agents in conjunction with the polypeptides of the present invention.
  • conjugates are used in its broadest sense to encompass all methods of attachment or joining that are known in the art. Such conjugates include fusion proteins, those produced by chemical conjugates and those produced by any other methods.
  • conjugates include fusion proteins, those produced by chemical conjugates and those produced by any other methods.
  • conjugated is used interchangeably with terms such as “linked”, “bound”, “associated”, “fused” or “attached”.
  • a wide range of covalent and non-covalent forms of conjugation are known to the person of skill in the art, and fall within the scope of the invention. For example, disulphide bonds, chemical linkages and peptide chains are all forms of covalent linkages.
  • the means of attachment may be, for example, a biotin-(strept)avidin link or the like.
  • Antibody (or antibody fragment)- antigen interactions may also be suitably employed to conjugate a cyclotide of the invention to another moiety, such as a polypeptide, peptide, non-polypeptide moiety, small molecule, or biological drug.
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, "binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure.
  • Percent (%) amino acid sequence identity refers to the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MegAlign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • Amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, gamma-carboxy glutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an alpha carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs can have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions similarly to a naturally occurring amino acid.
  • amino acid variants refers to amino acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical or associated (e.g., naturally contiguous) sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
  • nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes silent variations of the nucleic acid.
  • AUG which is ordinarily the only codon for methionine
  • TGG which is ordinarily the only codon for tryptophan
  • nucleic acid which encodes a polypeptide is implicit in a described sequence with respect to the expression product, but not with respect to actual probe sequences.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" including where the alteration results in the substitution of an amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles disclosed herein.
  • conservative substitutions include: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
  • a “polypeptide” is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or in vitro by synthetic means.
  • a polymer of amino acids of 2 to 50 amino amino acids typically referred to as “peptides”.
  • the term “polypeptide” as used herein denotes the product of a naturally occurring polypeptide, precursor form or proprotein.
  • the term also appies to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers.
  • Polypeptides can also undergo maturation or post- translational modification processes that may include, but are not limited to: glycosylation, proteolytic cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, and such like.
  • the term “protein” is used herein to refer to a large polypeptide molecule as well as a macromolecule comprising one or more polypeptide chains.
  • peptide refers to a plurality of amino acids joined together, such as in a linear or circular chain.
  • oligopeptide is typically used to describe peptides having between 2 and about 50 or more amino acids. Peptides larger than about 50 are often referred to as polypeptides or proteins.
  • the person of skill in the art understands and can determine whether a polypeptide molecule is an polyprotein or peptide sequence, for example, by way of sequence homology or structure prediction or determination.
  • Such non-polypeptide moieties include nucleic acids and other polymers, peptides, proteins, peptide nucleic acids (PNAs), antibodies, antibody fragments, and small molecules, amongst others.
  • a non-polypeptide moiety is a biological molecule (e.g. comprising a polynucleotide or peptide), and advantageously is a therapeutic or targeting molecule.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, heavy chain variable (VH) regions capable of specifically binding, and single chain variable fragments (scFv).
  • Fab fragment antigen binding
  • rlgG fragment antigen binding
  • VH heavy chain variable
  • an “antibody fragment” comprises a portion of an intact antibody, the antigen binding and/or the variable region of the intact antibody.
  • Antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide- linked Fvs (dsFv), Fd fragments, Fd' fragments; single-chain antibody molecules, including single-chain Fvs (scFv) or single-chain Fabs (scFab); antigen-binding fragments of any of the above and multispecific antibodies from antibody fragments.
  • a “Fab fragment” is an antibody fragment that results from digestion of a full- length immunoglobulin with papain, or a fragment having the same structure that is produced synthetically, e.g., by recombinant methods.
  • a Fab fragment contains a light chain (containing a VL and CL) and another chain containing a variable domain of a heavy chain (VH) and one constant region domain of the heavy chain (CHI).
  • an “scFv fragment” refers to an antibody fragment that contains a variable light chain (VL) and variable heavy chain (VH), covalently connected by a polypeptide linker in any order.
  • the linker is of a length such that the two variable domains are bridged without substantial interference.
  • Exemplary linkers are (Gly-Ser) n residues with some Glu or Lys residues dispersed throughout to increase solubility.
  • operably linked when applied to DNA sequences, for example in an expression vector or construct indicates that the sequences are arranged so that they function cooperatively in order to achieve their intended purposes, i.e. a promoter sequence allows for initiation of transcription that proceeds through a linked coding sequence as far as the termination sequence.
  • nucleic acid sequence is a single or double stranded covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds.
  • the polynucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases.
  • Nucleic acid sequences may include DNA and RNA, and may be manufactured synthetically in vitro or isolated from natural sources.
  • polynucleotides are typically expressed as the number of base pairs (bp) for double stranded polynucleotides, or in the case of single stranded polynucleotides as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides of less than around 40 nucleotides in length are typically called “oligonucleotides” and may comprise primers for use in manipulation of DNA such as via polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • vector is used to denote a DNA molecule that is either linear or circular, into which another nucleic acid (typically DNA) sequence fragment of appropriate size can be integrated.
  • DNA fragment(s) can include additional segments that provide for transcription of a gene encoded by the DNA sequence fragment.
  • additional segments can include and are not limited to: promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selectable markers, origins of replication and such like.
  • promoters e.g.
  • an expression vector that encodes a modified polypeptide or fragment/ domain thereof that comprise a molecule of the invention.
  • the DNA encoding a relevant peptide of the invention can be inserted into a suitable expression vector (e.g. pGEM®, Promega Corp., USA), where it is operably linked to appropriate expression sequences, and transformed into a suitable host cell for protein expression according to conventional techniques (Sambrook J. et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY).
  • Suitable host cells are those that can be grown in culture and are amenable to transformation with exogenous DNA, including bacteria, fungal cells and cells of higher eukaryotic origin, preferably mammalian cells, typically human cells and cell lines.
  • the polypeptide (and corresponding nucleic acid) of the invention may include a purification sequence, such as a His-tag.
  • the modified polypeptides may, for example, be grown in fusion with another protein and purified as insoluble inclusion bodies from bacterial cells. This is particularly convenient when the modified polypeptide to be synthesised may be toxic to the host cell in which it is to be expressed.
  • modified polypeptides may be synthesised in vitro using a suitable in vitro (transcription and) translation system (e.g. the E. coli S30 extract system: Promega corp., USA).
  • the vector is suitable as a polypeptide library display vector, enabling the polypeptide gene product of the cyclotide encoding gene to remain associated with the vector following transcription.
  • polypeptide library display refers to a system in which a collection of polypeptides or peptides, that may form part or all of a library, are made available for selection based upon a specified characteristic.
  • the specified characteristic may be a physical, chemical or functional characteristic.
  • Suitable display systems utilise a cellular expression system, for instance an expression of a library of nucleic acids in appropriately transformed, infected, transfected or transduced cells and display of the encoded polypeptides on the surface of the cells.
  • Alternative cellular expression systems may include emulsion compartmentalization and display.
  • Optional display systems link the coding function of a nucleic acid and physical, chemical and/or functional characteristics of a polypeptide or peptide encoded by the nucleic acid.
  • polypeptides or peptides that have a desired physical, chemical and/or functional characteristic can be selected and the nucleic acid encoding the selected polypeptide is readily isolated.
  • display systems that link the coding functionality of a nucleic acid with the associated polypeptide product are known in the art, for example, bacteriophage display (phage display), ribosome display, emulsion compartmentalization and display, yeast display, puromycin display, bacterial display, display on plasmid, covalent display, CIS display and the like.
  • the term "library” refers to a mixture of heterogeneous polypeptides or nucleic acids.
  • the library is composed of a plurality of members, each of which has a substantially unique polypeptide or nucleic acid sequence. Sequence differences between library members are responsible for the diversity present in the library.
  • the library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form of organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic acids.
  • each individual organism (such as a phage) or cell contains only one or a very limited number of library members.
  • a library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in nucleic acid form which can be expressed to produce its corresponding polypeptide member - i.e. the polypeptide gene product.
  • the population of host organisms has the potential to encode a widely diverse number of polypeptides.
  • An embodiment of the present invention provides for a library of polypeptides that are based around modified versions of cyclobody polypeptides, in which the diversity or variance between library members is located in the polypeptide sequences of one or more of the loop or variable regions of one or more functional modules within the protein.
  • a derivative of a cyclotide also is referred to as a “modified cyclotide.”
  • a derivative of a cyclotide polypeptide may contain one or more (e.g. 1, 2, 3, 4, 5 or more) amino acid mutations, substitutions or deletions to the primary sequence of a selected modified polypeptide.
  • the invention encompasses the results of maturation experiments conducted on a cyclotide polypeptide to improve or alter one or more characteristics of the initial or unmodified cyclotide.
  • one or more amino acid residues of a selected cyclotide polypeptide sequence may be randomly or specifically mutated (or substituted) using procedures known in the art (e.g. by modifying the encoding DNA or RNA sequence).
  • the resultant library or population of derivatised polypeptides may be selected - by any known method in the art - according to predetermined requirements: such as improved specificity against particular target receptors (e.g. receptors involved in transcytosis); improved transcytosis across the blood-brain barrier; or improved drug properties (e.g. solubility, bioavailability, immunogenicity etc.).
  • isolated with reference to binding molecules, such as peptides or antibodies or other binding molecules, or polynucleotides and vectors encoding same, are at least partially free of other biological molecules from the cells or cell culture from which they are produced.
  • biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other material such as cellular debris and growth medium.
  • An isolated binding molecule may further be at least partially free of expression system components such as biological molecules from a host cell or of the growth medium thereof.
  • isolated is not intended to refer to a complete absence of such biological molecules or to an absence of water, buffers, or salts or to components of a pharmaceutical formulation that includes the antibodies or fragments.
  • an effective amount or “therapeutically effective amount” as used herein means an amount of a pharmaceutical composition which is sufficient enough to significantly and positively modify the symptoms and/or conditions to be treated (e.g., provide a positive clinical response).
  • the effective amount of an active ingredient for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable excipient(s) and/or carrier(s) utilized, and like factors with the knowledge and expertise of the attending physician.
  • the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • the term “pharmaceutical composition” refers to a mixture of at least one compound of the invention with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
  • disease or disorder refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, genetic conditions, and characterized by identifiable symptoms.
  • the terms “treat,” “treating,” or “treatment” refer to ameliorating a disease or disorder, e.g., slowing or arresting or reducing the development of the disease or disorder, e.g., a root cause of the disorder or at least one of the clinical symptoms thereof.
  • the term "subject” refers to an animal, including a mammal (e.g., rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit), such as a human being.
  • a mammal e.g., rat, mouse, cat, dog, cow, pig, sheep, horse, goat, rabbit
  • the subject is a human subject.
  • subject and patient can be used interchangeably.
  • a modified cyclotide comprising: i) a peptide that binds to a blood-brain barrier trancytosis receptor (BBB-R) selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 Receptor Tyrosine Kinase 3 (ErbB3), leptin receptor (ObR), low- density lipoprotein receptor-related protein 1 (LRP-1), and receptor for advanced glycationend products (RAGE), wherein said peptide has an amino acid sequence of 2 to 50 amino acid residues; and ii) a cyclotide scaffold comprising the peptide of i), wherein the modified cyclotide comprises the structure (I): )
  • Ci to Ce are cysteine residues; wherein each of Ci and C 4 , C 2 and Cs, and C 3 and Ce are connected by a disulfide bond to form a cysteine knot; wherein each X represents an amino acid residue in a loop, wherein said amino acid residues are the same or different; wherein d is about 1-2; wherein at least one loop from loops 1, 2, 3, 5 or 6 has an amino acid sequence comprising the sequence of said peptide of clause i), wherein any loop comprising said sequence of said peptide of clause i) comprises 2 to about 30 amino acids, and wherein for any of loops 1, 2, 3, 5, or 6 that do not contain said sequence of said peptide of clause i), a, b, c, e, and f, are the same or different, and are each any number from 3-10, and b, c, e, and f are each any number from 1 to 20.
  • cyclotide scaffold is a Momordica cochinchinensis trypsin inhibitor.
  • Momordica cochinchinensis trypsin inhibitor is MCoTI-I set forth in SEQ ID NO: 1, MCoTI-II set forth in SEQ ID NO: 2 or MCoTI-III set forth in SEQ ID NO: 3.
  • a modified cyclotide comprising a peptide that binds to a blood-brain barrier trancytosis receptor (BBB-R), wherein: the peptide is inserted into or replaces one or more amino acids of at least one loop of the cyclotide scaffold set forth in SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO: 3, and wherein the peptide is about 2 to 50 amino acid residues; and the BBB-R is selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 Receptor Tyrosine Kinase 3 (ErbB3), leptin receptor (ObR), low-density lipoprotein receptor-related protein 1 (LRP-1), and
  • a modified cyclotide comprising a peptide inserted into loop 1 to replace all amino acids between cysteine 4 and cysteine 11 of SEQ ID NO:2, wherein the peptide is 2 to 50 amino acid residues and binds to a blood-brain barrier trancytosis receptor (BBB-R) selected from the group consisting of transferrin receptor (TrfR), insulin-like growth factor type 1 receptor (IGFR), Erb-B2 Receptor Tyrosine Kinase 3 (ErbB3), leptin receptor (ObR), low-density lipoprotein receptor-related protein 1 (LRP-1).
  • BBB-R blood-brain barrier trancytosis receptor
  • TrfR transferrin receptor
  • IGFR insulin-like growth factor type 1 receptor
  • ErbB3 Erb-B2 Receptor Tyrosine Kinase 3
  • ObR leptin receptor
  • LRP-1 low-density lipoprotein receptor-related protein 1
  • modified cyclotide of any of embodiments 1-16 wherein the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2 to 20 amino acids, 2 to 15 amino acids, 2 to 10 amino acids, 2 to 5 amino acids, 5 to 50 amino acids, 5 to 40 amino acids, 5 to 30 amino acids, 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 5 to 10 amino acids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids, 10 to 25 amino acids, 10 to 15 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 15 to 30 amino acids, 15 to 25 amino acids, 15 to 20 amino acids, 20 to 50 amino acids, 20 to 40 amino acids, 20 to 30 amino acids, 20 to 25 amino acids, 25 to 50 amino acids, 25 to 40 aminio acids, 25 to 30 amino acids, 30 to 50 amino acids, 30 to 40 amino acids, or 40 to 50 amino acids.
  • the peptide is 2 to 40 amino acids, 2 to 30 amino acids, 2 to 25 amino acids, 2
  • amino acids such as 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids.
  • modified cyclotide of any of embodiments 1-23 and 34-36 wherein the modified cyclotide comprises the sequence set forth in any one of SEQ ID NOS: 56-69, optionally wherein the modified cyclotide is set forth in any one of SEQ ID NOS: 56-69.
  • modified cyclotide of any of embodiments 1-23 and 39-41 wherein the modified cyclotide comprises the sequence set forth SEQ ID NO: 70 or 71, optionally wherein the modified cyclotide is set forth in SEQ ID NO: 70 or 71.
  • a peptide comprising the amino acid sequence set forth in any of SEQ ID NOs: 10-55 or 117-128, wherein the peptide is 6-50 amino acids in length and binds to a receptor involved in blood-brain barrier transcytosis (BBB-R).
  • BBB-R blood-brain barrier transcytosis
  • 2 to 30 amino acids such as 2 to 24 amino acids, 2 to 18 amino acids, 2 to 12 amino acids, 2 to 6 amino acids, 6 to 30 amino acids, 6 to 24 amino acids, 6 to 18 amino acids, 6 to 12 amino acids, 12 to 30 amino acids, 12 to 24 amino acids, 12 to 18 amino acids, 18 to 30 amino acids, 18 to 24 amino acids or 24 to 30 amino acids.
  • LRP-1 lipoprotein receptor-related protein 1
  • binding molecule of embodiment 82, wherein the binding scaffold is a cyclotide.
  • binding molecule of embodiment 83 or embodiment 84, wherein the binding scaffold is set forth in any one of SEQ ID NOS: 1-3.
  • binding molecule of any of embodiments 83-85, wherein the binding scaffold is set forth in SEQ ID NO:2.
  • a vector comprising the nucleic acid molecule of embodiment 87.
  • the vector of embodiment 88 that is an expression vector.
  • a host cell comprising the nucleic acid molecule of embodiment 87 or the vector of embodiment 87 or embodiment 89.
  • a method of producing a modified cyclotide or binding molecule comprising introducing the nucleic acid of embodiment 87 or the vector of embodiment 88 or embodiment 89 into a host cell and culturing the host cell under conditions to express the protein in the cell, optionally purifying the protein from the cell.
  • a conjugate comprising: a modified cyclotide of any of embodiments 1-60 or binding molecule of any of embodiments 82-86, and a biologically active agent.
  • a fusion protein comprising a modified cyclotide of any of embodiments 1-60 or binding molecule of any of embodiments 82-86, and a biologically active protein agent.
  • NGF nerve growth factor
  • GCSF Granulocyte colony-stimulating factor
  • conjugate or fusion protein of embodiment 102 wherein the enzyme is a ceramide degrading enzyme, a lipase, a hydrolase type enzyme or a sulfatase.
  • conjugate or fusion protein of embodiment 102 or 103 wherein the enzyme is a ceramide degrading enzyme and the ceramide degrading enzyme is glucocerebrosidase, galactocerebrosidase or alpha galactosidase.
  • conjugate or fusion protein of any of embodiments 93-105 comprising 2, 3, or 4 modified cyclotides.
  • conjugate or fusion protein of any of embodiments 93-107 and 108 that is monovalent for binding a BBB-R.
  • conjugate or fusion protein of embodiment 111 that is bispecific for binding two different BBB-R.
  • linker is a flexible peptide linker, optionally comprising the sequence GGGGS (SEQ ID NO: 148), (GGGGS) 2 (SEQ ID NO: 154) or (GGGGS) 3 (SEQ ID NO: 154).
  • a pharmaceutical composition comprising the conjugate or fusion protein of any of embodiments 93-119, and a pharmaceutical carrier.
  • a method for transporting a biologically active agent across a blood brain barrier of an individual comprising administering the conjugate or fusion protein of any of embodiments 93-119 or the pharmaceutical composition of embodiment 120 to a mammal in need thereof.
  • invention 121 wherein the mammal has a neurological disease.
  • said neurological disease is selected from the group consisting of Alzheimer’s disease, Parkinson's disease, stroke, a brain tumor and a brain metastasis.
  • invention 124 The method of embodiment 121 or embodiment 123, wherein said neurological disease is a congenital disease that is selected from the group consisting of Austen Disease, Canavan Disease, Gaucher’s Disease, Hunter Syndrome, Hurler-Scheie Syndrome, Jansky Bielschowsky Disease, Krabbe Disease, LCAT Deficiency, Lowe Syndrome, Maroteaux-Lamy Syndrome, Morquio Syndrome A, Morquio Syndrome B, Sanfilippo Syndrome A, Sanfilippo Syndrome B, Sanfilippo Syndrome C, Sanfilippo Syndrome D, Spinal Muscular Atrophy, Tay Sachs Disease, and Walker-Warburg Syndrome.
  • a method for treating a patient having a neurological disease comprising administering the conjugate of any of embodiments 46-52 or the pharmaceutical composition of embodiment 53 to said patient.
  • a method for diagnosing a neurological disease in a patient in need thereof comprising administering the conjugate or fusion protein of any of embodiments 93- 119 or the pharmaceutical composition of embodiment 53 to said patient and wherein said conjugate comprises a radiolabel.
  • composition of embodiment 120 for use in the treatment of a neurological disease.
  • composition of embodiment 120 for use in the diagnosis of a neurological disease.
  • Cyclotide libraries were created by insertion of random peptides into the backbone cyclotide MCOTII scaffold set forth in SEQ ID NO:2.
  • the libraries were based on the MCOTII native DNA sequence (SEQ ID NO:4) as a cyclotide scaffold and were built containing mutant sequences between the first two cysteine residues of SEQ ID NO:2, (termed “Loopl”), in which the libraries differed in the number of randomized peptides.
  • oligonucleotides were designed to insert the mutant sequences into Loop 1 to generate a library with a loop of 10 amino acids (“LooplforlO”; SEQ ID NO:5) or 12 amino acids (“Looplforl2”, SEQ ID NO: 6) when expressed.
  • a further library was similarly generated to encode 14-20 randomized amino acids in Loopl. All randomised amino acid positions were encoded in the library to represent an equal mixture of codons for 19 of the naturally occurring amino acids excluding cysteine.
  • PCR products were cloned as Ncol-Notl digested fragments into similarly digested pSPl phagemid pill fusion vector derived from the pHENl pill vector (Hoogenboom et al., 1991, Nucleic Acids Res., 19: 4133-4137).
  • Each 50 pl reaction mixture contained 10 ng pCyclol, 25 pmol of the appropriate forward and reverse primers, 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and lx NEB PCR reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM (NH 4 ) 2 SO4, 10 mM KC1, 2 mM MgSO 4 , 0.1% Triton X-100; NEB Ltd, Cambridge, UK). Reactions were performed for 30 PCR cycles of 94°C, 20s; 60°C, 40s; 72°C, 30s, followed by 5 minutes at 72°C. Reaction products were purified using two Wizard PCR clean-up columns per repertoire (Promega Ltd, Southampton, UK), and eluted into 50 pl water per column.
  • Each 50 pl reaction mixture contained approximately 25 ng primary Cyclotide Loop library, 25 pmol of the appropriate forward and reverse primers, 0.1 mM dNTPs, 2.5 units Taq DNA polymerase, and lx NEB PCR reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM (NH 4 )2SO 4 ,10 mM KC1, 2 mM MgSO 4 , 0.1% Triton X-100; NEB Ltd, Cambridge, UK). Reactions were performed for 25 cycles of 94°C, 20s; 60°C, 40s; 72°C, 30s, followed by 5 minutes at 72°C. Reaction products were purified using four Wizard PCR clean-up columns per library (Promega Ltd, Southampton, UK), and eluted into 100 pl water per column.
  • Each of the libraries, and 250 pg pSPl vector DNA were digested with enzymes Ncol and Noil (100 units each enzyme) for 5 hours at 37°C (NEB, Cambridge, UK), and purified using one Wizard PCR clean-up column per library, and four Wizard PCR clean-up columns for the digested vector DNA (Promega Ltd, Southampton, UK). Each DNA sample was then eluted into 100 pl water. Half of each digested library DNA was ligated overnight at 16°C in 400 pl with 50 pg of Ncol-Notl cut pSPl vector and 4000U of T4 DNA ligase (NEB Ltd,shire, UK).
  • the ligations were adjusted to 200 pl with nuclease free water, and DNA precipitated with 1 pl 20 mg/ml glycogen, 100 pl 7.5M ammonium acetate and 900 pl ice-cold (-20°C) absolute ethanol, vortex mixed and spun at 13,000 RPM for 20 minutes in a microfuge to pellet DNA.
  • the pellets were washed with 500 pl ice-cold 70% ethanol by centrifugation at 13,000 RPM for 2 minutes, then vacuum dried and re-suspended in 100 pl DEPC-treated water. 1 pl aliquots of each library were electroporated into 80 pl E. coll (TGI).
  • Bacterial cells were grown in 1 ml SOC medium per cuvette for 1 hour at 37°C, and plated onto 2x TY agar plates supplemented with 2% glucose and 100 pg/ml ampicillin. 10' 4 , 10' 5 and 10' 6 dilutions of the electroporated bacteria were also plated to assess library size. Colonies were allowed to grow overnight at 30°C. Combined library size was approximately 2x 10 10 clones with >95% with in-frame inserts.
  • Infected bacteria were transferred to 200 ml 2x TY broth supplemented with 25 pg/ml kanamycin, 100 pg/ml ampicillin, and 20 pM IPTG, then incubated overnight at 30°C, shaking at 200 RPM. Bacteria were pelleted at 4000 RPM for 20 minutes in 50 ml Falcon tubes, and 40 ml 2.5 M NaCl / 20% PEG 6000 was added to 400 ml of particle supernatant, mixed vigorously and incubated on ice for 1 hour to precipitate phage particles.
  • Particles were pelleted at 11000 RPM for 30 minutes in 250 ml Oakridge tubes at 4°C in a Sorvall RC5B centrifuge, then resuspended in 40 ml water and 8 ml 2.5M NaCl / 20% PEG 6000 added to reprecipitate particles, then incubated on ice for 20 minutes. Particles were again pelleted at 11000 RPM for 30 minutes in 50 ml Oakridge tubes at 4°C in a Sorvall RC5B centrifuge, then resuspended in 5 ml PBS buffer, after removing all traces of PEG / NaCl with a pipette.
  • Bacterial debris was removed via centrifugation for 5 minutes at 13500 RPM in a microcentrifuge. The supernatant was filtered through a 0.45 pm polysulfone syringe filter, adjusted to 20% glycerol and stored at - 70°C.
  • Cyclotide phage libraries described in Example 1 were screened for receptor binding peptides able to bind a receptor capable of inducing receptor-mediating transcytosis (RMT) across the blood-brain barrier (BBB), including human HER3ZErbB3 (Hu ErbB3), human and mouse transferrin receptor (Hu TrfR or Mu TrfR), human leptin receptor (Hu ObR), human insulin-like growth factor type 1 receptor (Hu IGFR), human low-density lipoprotein receptor-related protein 1 (Hu LRP-1), and human receptor for advanced glycation-end products (Hu RAGE).
  • RTT receptor-mediating transcytosis
  • TfR cyclotide selection For TfR cyclotide selection, a round of selection was also carried out in the presence of human holo-transferrin, such that selections were carried out against human TfR when human holo-transferrin is bound to the receptor. In this selection, CYC40 (SEQ ID NO:95) was the most predominant peptide hit in this selection.
  • Table El sets forth sequences of selected cyclotides and the receptors they bind. Table El. Cyclotide and receptor binding peptide target and sequences
  • adalimumab-cyclotide fusion was purified by Protein A affinity chromatography and concentrated. Two exemplary different adalimiumab-cyclotide fusion proteins showed no signs of enhanced aggregation when compared with the non-cyclotide fused version of the antibody as determined by western blot (data not shown).
  • the exemplary cyclotide CYC17 (also called HT2; set forth in SEQ ID NO:72), which binds the human transferrin receptor, was fused to adalimumab.
  • the adalimumab- CYC17 cyclotide fusion was compared to adalimumab for internalization via the transferrin receptor.
  • the CYC17 cyclotide set forth in SEQ ID NO:72 was linked to the C-terminal end of the heavy chain (SEQ ID NO: 102) of adalimumab via a Gly-Ser linker (e.g., SEQ ID NO: 104).
  • a genetic construct for expression of the adalimumab -CYC 17 cyclotide fusion protein was prepared by cloning the cyclotide gene fused to the 3’ end of a nucleotide sequence coding for the antibody heavy chain gene (with 5’ IL2 secretory signal peptide sequence) into a mammalian expression vector.
  • adalimumab heavy chain-CYC17 cyclotide fusion protein or adalimumab heavy chain alone
  • the adalimumab light chain amino acid sequences set forth in SEQ ID NO: 103
  • HEK293F cells were grown for 4 to 6 days at 37°C, 8% CO2, 130 rpm shaking.
  • the resulting adalimumab-CYC17 cyclotide fusion protein was purified by Protein A affinity chromatography and concentrated using a 50 KDa molecular weight cutoff centrifugal filtration unit (Amicon).
  • adalimumab and adalimumab-CYC17 cyclotide fusion proteins were fluorescently labeled by incubation with a 5- to 10-fold molar excess of Alexafluor 488 N- hydroxysuccinamide (NHS) ester for one hour at room temperature, pH ⁇ 8.3 in the dark. Free dye was then removed by passage through one to two desalting columns.
  • NHS N- hydroxysuccinamide
  • HEK293F cells were first serially passaged two to three times, then transfected with expression constructs encoding human or mouse transferrin receptor (hTfR, mTfR, respectively), and grown for 2-3 days at 37°C, 8% CO2, 130 rpm shaking in preparation for the assay.
  • hTfR human or mouse transferrin receptor
  • each adalimumab heavy chain-mutant CYC 17 cyclotide fusion and the adalimumab light chain were co-transfected into HEK293F cells, which were grown for 4 to 6 days at 37°C, 8% CO2, 130 rpm shaking. After removal of cells and filtration of the supernatant, the resulting adalimumab-cyclotide fusion was purified by Protein A affinity chromatography and concentrated.
  • Exemplary CYC17 mutants are set forth in Table E2. Binding changes to human transferrin receptor for the exemplary CYC 17 mutants were assessed by ELISA.
  • TrFR His-tagged human Transferrin receptor
  • the heavy chain of the antibody aducanumab (AduhelmTM; SEQ ID NO: 129) was engineered by mutation of the CH3 to contain either a knob mutation (K; heavy chain-K set forth in SEQ ID NO: 130) mutation or a hole mutation (H; heavy chain-H set forth in SEQ ID NO: 131).
  • the heavy chain-K of aducanumab was fused to cyclotide CYC27 (SEQ ID NO:82) for IGFIR-targeting.
  • the heavy chain-K or the heavy chain-H of aducanumab was fused to cyclotide CYC 17 (SEQ ID NO: 72) for hTfR-targeting.
  • Nucleic acids encoding the heavy chain-K and heavy -H fusions were introduced with the nucleic acid encoding the light chain of aducanumab (SEQ ID NO: 132) into HEK293F for expression and production of monovalent and bispecific heterodimeric aducanumab-cyclotide fusions.
  • SEQ ID NO: 132 The nucleic acid encoding the light chain of aducanumab
  • the heavy chain of the antibody adalimumab (HumiraTM; SEQ ID NO: 102) was fused to cyclotide CYC 17 for hTfR-targeting as described above.
  • the expression constructs for the adalimumab heavy chain-CYC17 cyclotide fusion and the adalimumab light chain (SEQ ID NO: 103) were used to co-transfect HEK293F cells for expression and production of a bivalent adalimumab-cyclotide fusion.
  • a schematic of the bivalent adalimumab-CYC17 cyclotide fusion is depicted in FIG. 1A.
  • cleavable cathepsin B cleavage site between the linker and cyclotide was added to promote cleavage of the cyclotide peptide portion from the fusion protein after its internalization into the endosome to enhance antibody release from the endosome and transport of the antibody across the blood-brain barrier.
  • the heavy chain of the antibody trastuzumab (Herceptin®, SEQ ID NO: 134) was engineered by mutation of the CH3 to contain either a knob mutation (K; heavy chain-K set forth in SEQ ID NO: 135) or a hole mutation (H; heavy chain-H set forth in SEQ ID NO: 136).
  • the heavy chain-K or the heavy chain-H of trastuzumab was fused to cyclotide CYC17 (SEQ ID NO:72) or the lower affinity mutant CYC17-15H (SEQ ID NO: 101) for human transferrin receptor (hTfR)-targeting.
  • the cyclotide was linked to the C- terminal end of the heavy chain-K (SEQ ID NO: 135) of trastuzumab via a Gly-Ser linker (e.g. SEQ ID NO: 104), and, in some cases further including the cathepsin B cleavage sequence (SEQ ID NO: 133).
  • a Gly-Ser linker e.g. SEQ ID NO: 104
  • the heavy chain-K of trastuzumab was fused to cyclotide CYC27 (SEQ ID NO:82) for IGFIR-targeting.
  • Table E4 sets forth the sequence of generated constructs.
  • Nucleic acids encoding the respective full heavy chain-K (e.g., SEQ ID NOS: 139, 140, 141 or 142) and full heavy-H fusions (e.g., SEQ ID NOS: 136 or 143) were introduced with the nucleic acid encoding the light chain of trastuzumab (SEQ ID NO: 137) into HEK293F for expression and production of monovalent and bispecific heterodimeric trastuzumab-cyclotide fusions.
  • trastuzumab control nucleic acids encoding the wildtype (WT) trastuzumab heavy chain (SEQ D NO: 134) and the light chain of trastuzumab (SEQ ID NO: 137) were introduced into HEK293F cells. Cells were grown for 4 to 6 days at 37°C, 8% CO2, 130 rpm shaking. After removal of cells and filtration of the supernatant, the resulting trastuzumab-cyclotide fusion proteins were purified by Protein A affinity chromatography and concentrated.
  • Results for expression of exemplary constructs is shown in FIG. 5.
  • Trastuzumab fusion proteins (C, D, E and F in Table E4) expressed at similar levels to the parental antibody (A in Table E4) as determined by ELISA, with no evidence of aggregation by SDS- PAGE gel electrophoresis.
  • the exemplary human transferrin receptor (hTfR) binding cyclotide CYC17 (SEQ ID NO:72) was fused to the enzyme glucocerebrosidase (GlcCSase; SEQ ID NO: 144), an enzyme that cleaves by hydrolysis the beta-glucosidic linkage of the chemical glucocerebroside.
  • the cyclotide was linked to the C-terminal end of glucocerebrosidase via a Gly-Ser linker (e.g. SEQ ID NO: 104).
  • a standard glucocerebroside activity assay based on the ability of the GlcCSase- CYC17 fusion cyclotide to react with substrate was performed to assess the functional integrity/activity of the fusion cyclotide. Specifically, the enzymatic activity was assayed using the SensoLyte Red Glucocerebrosidase Fluorometric Assay Kit (AnaSpec) according to the manufacturer’s protocol. The assay uses a resorufin-P-glucoside substrate that, upon cleavage of the glycosidic linkage by hGCSase, releases resorufin, which fluoresces at 610 nm upon excitation at 570 nm.
  • the kinetic assay was conducted in triplicate in an opaque 96- well plate at room temperature in a SpectraMax fluorescence plate reader over the course of 1 hour using 10 pL of filtered, concentrated supernatant from the HEK293F hGCSase-CYC17 expression culture, 40 pL of assay buffer, and 50 pL of substrate solution. Positive (GCSase enzyme), inhibitor, vehicle only, and substrate only controls were assayed parallelly in triplicate.
  • Binding of the GlcCSase-CYC17 fusion cyclotide was assessed by Enzyme-linked immunosorbent assay (ELISA) of hGCSase-HT2 using human transferrin receptor (hTfR) antigen.
  • ELISA Enzyme-linked immunosorbent assay
  • Duplicate wells of a high binding half area 96-well plate (Coming) were directly coated with 125 ng of hTfR in 50 pL Tris buffered saline plus 0.1% (v/v) Tween 20 (TBST) containing 2% milk powder (or TBST containing 2% milk powder alone as a negative control) at 4°C overnight.
  • the wells are blocked with 50 pL of TBST containing 2% milk powder at room temperature for 2 hours.
  • the blocking agent was removed, and 25 pL of filtered, concentrated supernatant from the HEK293F hGCSase- CYC17 fusion expression culture (or 25 pL of filtered, concentrated supernatant from a non- hGCSase expressing HEK293F expression culture as a negative control) plus 25 pL of TBST containing 4% milk powder were added to the appropriate wells and incubated at room temperature for 1 hour. The supernatants were removed, and wells were washed 4 times with TBST.
  • a ⁇ 1 mg/mL rabbit anti-GCSase primary antibody (Sigma) was diluted 1 :300 in TBST containing 2% milk powder, 50 pL is added to each well, and the plate was incubated a room temperature for 1 hour. The supernatants were removed, and the wells were washed 4 times with TBST.
  • a ⁇ 1 mg/mL goat anti-rabbit IgG-HRP secondary antibody was diluted 1 :5,000 in TBST containing 2% milk powder, 50 pL is added to each well, and the plate was incubated a room temperature for 30 minutes. The supernatants were removed, and wells were washed 4 times with TBST followed by one time with Tris buffered saline (TBS).
  • TMB tetramethylbenzidine
  • the exemplary human transferrin receptor (hTfR) binding cyclotide CYC17 (SEQ ID NO:72) was fused to the C-terminal end of nerve growth factor (NGF).
  • NGF nerve growth factor
  • Genetic constructs for expression of therapeutic human NGF-cyclotide fusions were prepared by cloning the cyclotide gene fused to the 3’ end of the human Pro-NGF gene (encoding an amino acid sequence set forth in SEQ ID NO: 147) via a sequence encoding a flexible linker.
  • the construct included a 5’ IL2 secretory signal peptide sequence for expression.
  • the sequence of the Pro-NGF-CYC17 fusion construct encoded an amino acid sequence set forth in SEQ ID NO: 149.
  • the genetic construct also included a nucleotide sequence that encoded for a V5 epitope tag (SEQ ID NO: 150) and 6-His tag (SEQ ID NO: 151) at the 3’-end of the cyclotide.
  • the genetic construct was cloned into a mammalian expression vector.
  • An exemplary nucleic acid sequence encoding the NGF-CYC17 fusion cyclotide sequence is set forth in SEQ ID NO: 153.
  • T75 flasks were seeded with 15 mL of HEK 293 EBNA cells at 8 x 10 5 cells/ml in Optimem medium (Invitrogen) and incubated over night at 37°C in a humidified incubator supplemented with 5% CO2.
  • 180 pL of purified plasmid DNA encoding the cyclotide fusion at l.Omg/mL in sterile water was added to 600 pL of Optimem.
  • 66 pL Lipofectamine 2000 (Invitrogen) was added to 660 pL Optimem, mixed briefly and incubated at room temperature for 5 minutes. The two solutions were then combined and incubated for a further 20 minutes at room temperature.
  • Bound proteins were then eluted via the addition of 0.5 mL PBS supplemented with 250 mM imidazole. Imidazole was removed via dialysis in 2 L PBS (pH 7.4) overnight at room temperature, using Slidealyzer dialysis cassettes (10,000 MW cut-off) (Thermo Fisher Scientific Ltd).
  • NGF-cyclotide fusion was contacted with His-tagged human transferrin receptor (TrFR) coated at various concentrations (0.5 pg/mL, 1 pg/mL, or 2 pg/mL) on a microwell plate. Binding of the NGF-cyclotide fusion to the coated human transferrin receptor was assessed by ELISA by anti-V5-HRP detection of the NGF-CYC17 fusion protein. As shown in FIG. 7, the NGF-cyclotide fusion bound to hTrFR.
  • TrFR His-tagged human transferrin receptor
  • Table E5 sets forth the exemplary tested cyclotide fusions and trastuzumab control. Among the tested cyclotide fusions were fusion conjugates that exhibited different binding affinities for the BBB-R.
  • heterodimeric cyclotide trastuzumab fusions of CYC30 (SEQ ID NO:85), CYC32 (SEQ ID NO:82) and CYC34 (SEQ ID NO: 89) were assessed for binding affinity to human LRP1 by ELISA.
  • the IC50 for binding h LPR1 was 19.25 nM for CYC30 antibody fusion, 73.69 nM for CYC34 antibody fusion, and 830.2 nM for CYC32 antibody fusion.
  • mice Female C57BL/6 mice, age 10-12 weeks, were weighed and administered purified trastuzumab antibody or trastuzumab antibody-monovalent cyclotide fusions, made monovalent by knobs-holes fusion (targeting IGF1R, LRP1, or RAGE receptors) in triplicate at 25 mg/kg in phosphate buffered saline (PBS). After 24 hours, mice were perfused with Dulbecco’s PBS containing anesthetic via aortic puncture and brains were removed.
  • PBS phosphate buffered saline
  • One brain hemisphere was transferred to 4% paraformaldehyde in PBS for 24 hours at 4°C, then transferred to PBS at 4°C, paraffin embedded, thin-sectioned using a microtome, and slices were mounted on slides for immunohistochemistry (IHC).
  • the other brain hemisphere was flash frozen and stored at -80°C prior to homogenization.
  • Antibodies present in brain were quantified by enzyme-linked immunosorbent assay (ELISA). Clear half-area high binding 96-well plates were coated with 250 ng/well donkey anti-human Fc IgG overnight at 4°C (Jackson). Liquid was removed, wells were blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) containing 2% (w/v) milk powder at room temperature for 1 hour, and liquid was again removed. Neat and serially diluted clarified brain homogenates and serial dilutions of each antibody standard were prepared in TBST containing 2% (w/v) milk powder, added in duplicate to plates, and incubated for 1 hour at room temperature.
  • ELISA enzyme-linked immunosorbent assay
  • Amounts of antibodies e.g., antibody fusion proteins or antibody alone
  • Amounts of antibodies present in brain homogenates were determined by interpolation from each standard curve and were expressed as percents of injected dose per gram of brain (FIG. 8B).
  • Cyclotides targeting LRP1, IGF1R and RAGE enhanced trastuzumab delivery compared with unlabelled trastuzumab. These data demonstrate that cyclotide fusions targeting RMT receptors improve brain-specific delivery of a therapeutic agent.
  • mice Female C57BL/6 mice, age 10-12 weeks, were weighed and administered purified trastuzumab antibody or trastuzumab antibody-cyclotide fusions (monovalent CYC 17 or CYC17-15H mutant cyclotide paired in knobs-holes format) in triplicate at 25 mg/kg in phosphate buffered saline (PBS). After 24 hours mice were perfused with Dulbecco’s PBS containing anesthetic via aortic puncture and brains were removed.
  • PBS phosphate buffered saline
  • One brain hemisphere was transferred to 4% paraformaldehyde in PBS for 24 hours at 4°C, then transferred to PBS at 4°C, paraffin embedded, thin-sectioned using a microtome, and slices were mounted on slides for immunohistochemistry (IHC).
  • the other brain hemisphere was flash frozen and stored at -80°C prior to homogenization.
  • brain homogenization half brains were weighed and transferred to protease cocktail-treated bead beating homogenization tubes (Bertin Corp.). To each half brain was added 1 mL of PBS containing 1% NP-40, 5 mM EDTA; and tissue was disrupted using a bead beating homogenizer (Bertin Corp.) via 2 ⁇ 30 second pulses separated by a 15 second pause at 6,500 rpm. Brain homogenates were rotated at 4°C for 1 hour, then centrifuged at 14,000 rpm, 4°C for 20 minutes. Clarified homogenates (parenchyma) were carefully transferred to an Eppendorf tube and stored at -80°C.
  • Antibodies present in brain were quantified by enzyme-linked immunosorbent assay (ELISA). Clear half-area high binding 96-well plates were coated with 250 ng/well donkey anti-human Fc IgG overnight at 4°C (Jackson). Liquid was removed, wells were blocked with Tris-buffered saline containing 0.1% Tween 20 (TBST) containing 2% (w/v) milk powder at room temperature for 1 hour, and liquid was again removed. Neat and serially diluted clarified brain homogenates and serial dilutions of each antibody standard were prepared in TBST containing 2% (w/v) milk powder, added in duplicate to plates, and incubated for 1 hour at room temperature.
  • ELISA enzyme-linked immunosorbent assay

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Abstract

L'invention concerne des peptides capables de se lier à un récepteur qui médie la transcytose à médiation de récepteur (RMT) à travers la barrière hémato-encéphalique (BBB), ainsi que des molécules de liaison qui incorporent les peptides. L'invention concerne également des conjugués, comprenant des protéines de fusion, composés des peptides ou des molécules de liaison et d'un agent thérapeutique ou diagnostique. Dans certains modes de réalisation, les conjugués peuvent passer à travers la barrière hémato-encéphalique après avoir été administrés par voie parentérale pour activer la fonction de l'agent thérapeutique ou diagnostique dans le système nerveux central. L'invention concerne également des méthodes de fabrication et d'utilisation des peptides et molécules fournis.
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